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Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês

10.1074/jbc.m300276200

1083-351X

Laura Morelli, Ramiro E. Llovera, Silvia A. González, José L. Affranchino, Frances Prelli, Blas Frangione, Jorge Ghiso, Eduardo M. Castaño,

Cholinesterase and Neurodegenerative Diseases

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid β (Aβ) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Aβ wild type, yet whether IDE is capable of degrading Aβ bearing pathogenic substitutions is not known. We studied the degradation of all of the published Aβ genetic variants by recombinant rat IDE (rIDE). Monomeric Aβ wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Aβ Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Aβ wild type and the rest of the mutant peptides. In the case of Aβ Dutch, inefficient proteolysis was related to a high content of β structure as assessed by circular dichroism. All of the Aβ variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13–15 and 18–21. SDS-stable Aβ dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Aβ. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Aβ in hereditary forms of Aβ diseases. Inherited amino acid substitutions at position 21, 22, or 23 of amyloid β (Aβ) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Aβ wild type, yet whether IDE is capable of degrading Aβ bearing pathogenic substitutions is not known. We studied the degradation of all of the published Aβ genetic variants by recombinant rat IDE (rIDE). Monomeric Aβ wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Aβ Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Aβ wild type and the rest of the mutant peptides. In the case of Aβ Dutch, inefficient proteolysis was related to a high content of β structure as assessed by circular dichroism. All of the Aβ variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13–15 and 18–21. SDS-stable Aβ dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Aβ. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Aβ in hereditary forms of Aβ diseases. The accumulation of amyloid β peptide (Aβ) 1The abbreviations used are: Aβ, amyloid β; Aβ PP, amyloid β pre-cursor protein; IDE, insulin-degrading enzyme; rIDE, recombinant insulin-degrading enzyme; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type; GST, glutathione S-transferase; CD, circular dichroism; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HCHWA-D, hereditary cerebral hemorrhage with amyloidosis, Dutch type. 1The abbreviations used are: Aβ, amyloid β; Aβ PP, amyloid β pre-cursor protein; IDE, insulin-degrading enzyme; rIDE, recombinant insulin-degrading enzyme; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; WT, wild type; GST, glutathione S-transferase; CD, circular dichroism; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; HCHWA-D, hereditary cerebral hemorrhage with amyloidosis, Dutch type. in the brain is a central process in a number of human neurodegenerative disorders that may be grouped as "amyloid β diseases" (1Castaño E.M. Frangione B. Lab. Invest. 1988; 58: 122-132Google Scholar). In Alzheimer's disease, Aβ is mainly found within senile plaques in the neurophil and vascular lesions, whereas in sporadic and hereditary amyloid angiopathies, Aβ deposits are mainly associated with cortical and leptomeningeal vessels leading to stroke or multi-infarct dementia. To a lesser extent, cerebral Aβ deposits are also present in normal aging (2Coria F. Castaño E.M. Frangione B. Am. J. Pathol. 1987; 129: 422-428Google Scholar). Autosomal dominant mutations in the amyloid β precursor protein (Aβ PP) gene result in amino acid substitutions at position 21, 22, or 23 of Aβ sequence. Although these Aβ variants present with a primarily vascular deposition, they translate into different clinical phenotypes. In this regard, Aβ Arctic (E22G) and Aβ Iowa (D23N) are characterized by presenile dementia and Aβ Flemish (A21G) is associated with early onset dementia and cerebral hemorrhage, whereas Aβ Dutch (E22Q) and Aβ Italian (E22K) variants have a predominant vascular phenotype characterized by massive strokes (3Nilsberth C. Westlind-Danielsson A. Eckman C.B. Condron M.M. Axelman K. Forsell C. Stenh C. Luthman J. Teplow D.B. Younkin S.G. Naslund J. Lannfelt L. Nature Neurosci. 2001; 4: 887-893Google Scholar, 4Kamino K. Orr H.T. Payami H. Wijsman E.M. Alonso M.E. Pulst S.M. Anderson L. O'dahl S. Nemens E. White J.A. Sadovnick A.D. Ball M.J. Kaye J. Warren A. McInnis M. Antonarkis S.E. Korenberg J.R. Sharama V. Kukull W. Larson E. Heston L.L. Martin G.M. Bird T.D. Schellenberg G.D. Am. J. Hum. Genet. 1992; 51: 998-1014Google Scholar, 5Grabowski T.J. Cho H.S. Vonsattel J.P. Rebeck G.W. Greenberg S.M. Ann. Neurol. 2001; 49: 697-705Google Scholar, 6Miravalle L. Tokuda T. Chiarle R. Giaccone G. Bugiani O. Tagliavini F. Frangione B. Ghiso J. J. Biol. Chem. 2000; 275: 27110-27116Google Scholar, 7Levy E. Carman M.D. Fernandez-Madrid I.J. Power M.D. Lieberburg I. van Duinen S.G. Bots G.T. Luyendijk W. Frangione B. Science. 1990; 248: 1124-1126Google Scholar). The underlying mechanism of aggregation and deposition in vivo may be strongly influenced by the type of amino acid substitution as well as the location of mutations in the Aβ peptide. In vitro studies have shown that Aβ E22Q and Aβ D23N form typical amyloid fibrils at a higher rate than wild-type Aβ and that Aβ E22G assembles into unique protofibrils that may be toxic to neurons (3Nilsberth C. Westlind-Danielsson A. Eckman C.B. Condron M.M. Axelman K. Forsell C. Stenh C. Luthman J. Teplow D.B. Younkin S.G. Naslund J. Lannfelt L. Nature Neurosci. 2001; 4: 887-893Google Scholar, 8Wisniewski T. Ghiso J. Frangione B. Biochem. Biophys. Res. Commun. 1991; 173: 1247-1254Google Scholar, 9Castaño E.M. Prelli F. Wisniewski T. Golabek A. Kumar R.A. Soto C. Frangione B. Biochem. J. 1995; 306: 599-604Google Scholar, 10Van Nostrand W.E. Melchor J.P. Cho H.S. Greenberg S.M. Rebeck G.W. J. Biol. Chem. 2001; 276: 32860-32866Google Scholar). In the case of Aβ A21G, overproduction of the peptide may contribute as a pathogenic mechanism (11De Jonghe C. Zehr C. Yager D. Prada C.M. Younkin S. Hendriks L. Van Broeckhoven C. Eckman C.B. Neurobiol. Dis. 1998; 5: 281-286Google Scholar, 12Walsh D.M. Hartley D.M. Condron M.M. Selkoe D.J. Teplow D.B. Biochem. J. 2001; 355: 869-877Google Scholar). Regarding Aβ E22K, deposition seems not to be related with fibril formation rate, and yet this variant may be toxic to human cerebro-vascular smooth muscle cells in culture (6Miravalle L. Tokuda T. Chiarle R. Giaccone G. Bugiani O. Tagliavini F. Frangione B. Ghiso J. J. Biol. Chem. 2000; 275: 27110-27116Google Scholar, 13Melchor J.P. McVoy L. Van Nostrand W.E J. Neurochem. 2000; 74: 2209-2212Google Scholar). In addition to the intrinsic aggregation properties of Aβ and its genetic variants or the rate of their production, recent studies (14Gau J.T. Steinhilb M.L. Kao T.C. D'Amato C.J. Gaut J.R. Frey K.A. Turner R.S. Am. J. Pathol. 2002; 160: 731-738Google Scholar, 15Kuo Y.M. Beach T.G. Sue L.I. Scott S. Layne K.J. Kokjohn T.A. Kalback W.M. Luehrs D.C. Vishnivetskaya T.A. Abramowski D. Sturchler-Pierrat C. Staufenbiel M. Weller R.O. Roher A.E. Mol. Med. 2001; 7: 609-618Google Scholar) in animal models have suggested that a defective clearance may influence the progressive accumulation of Aβ in the neurophil and cerebral vessels. Among the mechanisms that remove Aβ peptides from the brain, degradation by several proteases including neprilysin, endothelin-converting enzyme, and insulin degrading enzyme (IDE) is now being considered as an important component of the Aβ clearance process (16Iwata N. Tsubuki S. Takaki Y. Shirotani K. Lu B. Gerard N.P. Gerard C. Hama E. Lee H.J. Saido T.C. Science. 2001; 292: 1550-1552Google Scholar, 17Eckman E.A. Reed D.K. Eckman C.B. J. Biol. Chem. 2001; 276: 24540-24548Google Scholar, reviewed in Refs. 18Selkoe D.J. Neuron. 2001; 32: 177-180Google Scholar and 19Carson J.A. Turner A.J. J. Neurochem. 2002; 81: 1-8Google Scholar). IDE is a highly conserved thiol metalloprotease with ubiquitous expression including the brain (20Baumeister H. Müller D. Rehbein M. Richter D. FEBS Lett. 1993; 317: 250-254Google Scholar, 21Kuo W. Montag A.G. Rosner M.R. Endocrinology. 1993; 132: 604-611Google Scholar, 22Becker A.B. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3835-3839Google Scholar). Regarding its physiological role, IDE has been implicated in cellular growth and differentiation, modulation of proteasomal activity, and steroid signaling (23Bennett R.G. Hamel F G. Duckworth W.C. Endocrinology. 2000; 141: 2508-2517Google Scholar, reviewed in Ref. 24Authier F. Posner B.I. Bergeron J.J.M. Clin. Invest. Med. 1996; 19: 149-158Google Scholar). In addition to insulin for which the protease has a Km in the low nanomolar range, IDE is known to degrade several peptides capable of forming amyloid fibrils in vitro and in vivo including glucagon, amylin, atrial natriuretic peptide, calcitonin, and Aβ (25Kurochkin I.V. Goto S. FEBS Lett. 1994; 345: 33-37Google Scholar, 26Bennett R.G. Duckworth W C. Hamel F.G. J. Biol. Chem. 2000; 275: 36621-36625Google Scholar, reviewed in Ref. 27Kurochkin I.V. Trends Biochem. Sci. 2001; 26: 421-425Google Scholar). Studies using rat and human brain tissue homogenates, IDE-transfected cell lines, and primary neuronal cultures have supported a role of IDE in Aβ degradation in vivo (28McDermott J.R. Gibson A.M. Neurochem. Res. 1997; 22: 49-56Google Scholar, 29Perez A. Morelli L. Cresto J.C. Castaño E.M. Neurochem. Res. 2000; 25: 247-255Google Scholar, 30Qiu W.Q. Walsh D.M. Ye Z. Vekrellis K. Zhang J. Podlisny M.B. Rosner M.R. Safavi A. Hersh L.B. Selkoe D.J. J. Biol. Chem. 1998; 273: 32730-32738Google Scholar, 31Gasparini L. Gouras G.K. Wang R. Gross R.S. Beal M.F. Greengard P. Xu H. J Neurosci. 2001; 21: 2561-2570Google Scholar). Moreover, IDE has been shown to protect cultured neurons from Aβ toxicity, indicating that the Aβ fragments generated by IDE are not themselves toxic to cells (32Mukherjee A. Song E. Kihiko-Ehmann M. Goodman J.P. St. Pyrek J. Estus E. Hersh L.B. J. Neurosci. 2000; 20: 8745-8749Google Scholar). Regarding the state of Aβ oligomerization and IDE specificity, it has been proposed that IDE is able to degrade efficiently monomeric Aβ as opposed to oligomeric Aβ species that are thought to be more toxic to neurons or vascular cells than typical amyloid fibrils (33Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D. Nature. 2002; 416: 535-539Google Scholar). However, whether IDE is capable of degrading Aβ bearing pathogenic amino acid substitutions and how these changes may affect specificity is not known. The aim of our work was to study the degradation of Aβ genetic variants associated with human disease by recombinant IDE in vitro and to characterize the proteolytic products from each of these Aβ mutant peptides.EXPERIMENTAL PROCEDURESExpression and Purification of Recombinant Rat IDE—The plasmid pECE-IDE (kindly provided by Richard Roth, Stanford University), containing the coding region of rat IDE cDNA was used as a template for the PCR amplification of a truncated version of IDE using sense 5′-AGCACAGGATCCATGAATAATCCGGCCAT-3′ (nucleotides 139–155) and antisense 5′-TTCTCGAGGAGTTTTGCCGCCATGA-3′ (nucleotides 3072–3056) primers of the rat IDE cDNA sequence. Sites for the restriction enzymes BamHI and XhoI, respectively, were introduced to facilitate cloning. The PCR DNA fragment was digested with BamHI and XhoI and cloned into pET-30a(+) (Novagen) previously digested with the same restriction enzymes to generate the pET-IDE construct. Recombinant IDE 42-1019 (rIDE) was expressed in Escherichia coli BL21 and purified to homogeneity using a Hi Trap Ni-chelating columm (Amersham Biosciences) following the method described by Chesneau and Rosner (34Chesneau V. Rosner M.R. Protein Expression Purif. 2000; 19: 91-98Google Scholar). Purity, as assessed by SDS-PAGE, was >95%. rIDE proteolytic activity was determined using [125I]insulin as described below.Production of Anti-IDE Polyclonal Antibodies—A region between amino acids 97 and 273 of the rat IDE sequence was amplified by PCR using as template pECE-IDE with the following primers: forward 5′-CTGAGCGGATCCCTGTCAGACCCTCCA-3′ and reverse 5′-CAATGTGAATTCCTTCACCACCAGATT-3′. After digestion with BamHI and EcoRI, the 530-bp insert was subcloned into pGex2T vector (Amersham Biosciences). GST-IDE97–273 fusion protein was expressed and purified according to the manufacturer's suggestions. After immunization of New Zealand rabbits with GST-IDE97–273, the antiserum (BC2) was sequentially purified using a BL21/GST lysate coupled to CNBr-activated Sepharose (Amersham Biosciences) and a GST-agarose (Sigma) affinity columns, respectively. Specificity of anti-IDE antiserum BC2 was tested by Western blot against GST, GST-IDE97–273, purified rIDE (see above), and soluble fractions from human and rat brain and liver in which a single 115-kDa band was detected (data not shown).Synthetic Peptides—Synthetic Aβ-(1–40)-peptides such as Aβ WT, Aβ A21G, Aβ E22Q, Aβ E22K, Aβ E22G, Aβ D23N, and Aβ-(1–40)/Aβ-(1–42) containing the rodent sequence (35Fraser P.E. Nguyen J.T. Inouye H. Surewicz W.K. Selkoe D.J. Podlisny M.B. Kirschner D.A. Biochemistry. 1992; 31: 10716-10723Google Scholar) were synthesized by the W. M. Keck Foundation (Yale University, CT). All of the peptides were purified by reverse-phase high pressure liquid chromatography, and their purity was evaluated by amino acid sequence analysis and laser desorption mass spectrometry. Lyophilized aliquots of the peptides were dissolved at a concentration of 70 μm in distilled water as determined with a bicinchoninic acid assay (Pierce) after centrifugation at 10,000 rpm for 15 min to eliminate large aggregates. The supernatant was aliquoted and stored at –80 °C.SDS-PAGE and Western Blot—Proteins obtained from E. coli BL21 lysates and after the different steps of purification were analyzed by 7.5% SDS-PAGE in Tris-Tricine gels. Aβ peptides were run on 12.5% Tris-Tricine SDS-PAGE. For Western blot analysis, proteins were transferred onto nitrocellulose membranes (Hybond ECL, Amersham Biosciences) and incubated with anti-Aβ monoclonal antibody 6E10 (Signet Laboratories) at 1:1000, anti-IDE monoclonal 9B12 (kindly provided by Richard Roth) at 1:1000, and polyclonal antibody BC2 at 1:1000. Immunoreactivity was detected with anti-mouse or anti-rabbit horseradish peroxidase-labeled IgG and ECL Plus (Amersham Biosciences). Immunoblots were scanned with STORM 840 and analyzed with ImageQuant 5.1 software (Amersham Biosciences).In-gel Tryptic Digestion of rIDE and Amino Acid Sequencing—After SDS-PAGE and Coomassie Blue staining, the band of 125 kDa was cut and the gel slice was incubated in 100 mm ammonium bicarbonate, pH 8.3 containing 45 mm dithiothreitol for 30 min at 60 °C. The tube was cooled at room temperature, and 100 mm iodoacetamide was added followed by incubation for 30 min in the dark at room temperature. The gel was then washed in 50% acetonitrile, 100 mm ammonium bicarbonate with shaking for 1 h, cut in pieces, and transferred to a small tube. Acetonitrile was added to shrink the gel pieces, and the sample was dried in a rotatory evaporator. The gel pieces were re-swollen with 10 μl of 100 mm ammonium bicarbonate, pH 8.3, containing trypsin at a 10:1 ratio (w/w, substrate:enzyme). The sample was incubated overnight at 37 °C, and digestion products were extracted twice from the gel with 60% acetonitrile, 0.1% trifluoroacetic acid for 20 min. Combined extractions were loaded into a C18 high pressure liquid chromatography column (220 × 1 mm), and peptides eluted with a linear gradient from 0 to 100% acetonitrile, 0.1% trifluoroacetic acid. Selected peaks were applied to a 477A protein-peptide sequencer (Applied Biosystems) and subjected to Edman degradation sequence analysis at the Laboratorio Nacional de Investigacíon y Servicios en Péptidos y Proteinas facility (CONICET).Degradation Assays—1 μg of each Aβ synthetic peptide was incubated alone or with 500 ng of purified rIDE in 10 μl of 100 mm sodium phosphate buffer, pH 7, in the presence or absence of 1 mm 1,10-phenantroline. After 30, 60, 90, and 120 min of incubation at 37 °C, samples were analyzed by SDS-PAGE and Western blot with 6E10 as described above. After each time point, degradation by rIDE was expressed as the percentage of remaining Aβ monomer or dimer as compared with Aβ incubated without the enzyme. [125I]Insulin (specific activity 300 μCi/μg, kindly provided by Edgardo Poskus, University of Buenos Aires.) was incubated with rIDE alone or in the presence of the following sets of inhibitors: 1) 1 μm unlabeled insulin; 2) a mixture of 2 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml pepstatin A; 3) a mixture of 1 mm 1,10-phenantroline and 5 mm EDTA, respectively, in the same buffer as above. After incubation for various time points, the sample was run on SDS-PAGE, and the remaining intact [125 I]insulin was analyzed and quantitated with a STORM 840 PhophorImager (Amersham Biosciences).Circular Dichroism (CD) Spectra—Aliquots of Aβ synthetic peptides freshly dissolved at 60 μm in 10 mm Tris-HCl, pH 7.5, were loaded into a 0.1-nm path length quartz cell, and the circular dichroism spectra were recorded in the far-ultraviolet light using a JASCO J-720 spectropolarimeter (JASCO Corporation). Forty scans for each experimental condition were obtained at 0.2-nm intervals over the wavelength range of 190–260 nm. Final spectra were obtained after the subtraction of background readings of blanks.Mass Spectrometry Analysis—Molecular masses of intact Aβ peptides and the products of rIDE degradation were determined at the New York University Protein Analysis Facility. Aβ-containing samples (10 μl of final volume) were passed through reversed-phase ZipTip (Millipore) following manufacturer's instructions and analyzed on a Micromass TofSpec-2E (MALDI-TOF) mass spectrometer in linear mode using standard instrument settings. Internal and/or external calibration was carried out using angiotensin I (average mass = 1296.5 Da) and insulin (average mass = 5733.5 Da).Isolation of Aβ from Leptomeningeal Vessels—Leptomeningeal vascular amyloid from a patient with hereditary cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D) was isolated as described previously (36Castaño E.M. Prelli F. Soto C. Beavis R. Matsubara E. Shoji M. Frangione B. J. Biol. Chem. 1996; 271: 32185-32191Google Scholar). Leptomeninges were dissected from brain coronal sections, cut with scissors into 1–3-mm pieces, and placed in 0.1 m Tris-HCl, pH 8, on ice. Tissue was then washed by resuspension in 0.1 m Tris-HCl, pH 8, containing protease inhibitors (buffer A) and centrifuged at 800 × g for 5 min at 4 °C. The procedure was repeated 5 times. The material was collected by filtration through a 50-μm nylon mesh (Spectrum), washed with buffer A without inhibitors, and resuspended in 20 volumes of 2 mm CaCl2 in 0.1 m Tris-HCl, pH 7.5, containing 0.3 mg/ml collagenase CLS-3 (Worthington) and 10 μg/ml DNase (Worthington). The mixture was incubated for 18 h at 37 °C. After digestion, the suspension was filtered through a 350-μm nylon mesh and the filtrate was centrifuged at 10,000 × g for 12 min. The pellet was resuspended in 2% SDS in 0.1 m Tris-HCl, pH 8, and incubated for 2 h at room temperature. The SDS-insoluble material was recovered by centrifugation as above and washed three times with distilled water. The pellet was dissolved in 10 volumes of 99% formic acid (Sigma) and incubated for 1 h at room temperature. After centrifugation at 10,000 × g for 10 min, the supernatant was then loaded on a Superose 12 column (10 × 300 mm) (Amersham Biosciences). Amyloid peptides were separated in 75% formic acid at 0.2 ml/min using a Bio-Cad Sprint chromatography system. Eluent was monitored at 280 nm, collected in 1-ml fractions, and analyzed as above.RESULTSExpression and Characterization of Recombinant Rat IDE— Our IDE bacterial expression vector pET-IDE encodes rat IDE (positions 42–1019) with a His6 tag at its N terminus to facilitate the purification of the protease with a Ni-affinity columm. After elution with a step gradient of increasing imidazole concentrations, we obtained a highly purified recombinant protein with an apparent molecular mass of 125 kDa as assessed by SDS-PAGE. Western blot using our polyclonal antibody BC2 specifically recognized this 125-kDa fusion protein (Fig. 1A). Unexpectedly, the protein did not display immunoreactivity on Western blot with monoclonal 9B12, a well characterized anti-rat IDE antibody (data not shown) (37Duckworth W.C. Hamel F.G. Bennett R. Ryan M.P. Roth RA. J. Biol. Chem. 1990; 265: 2984-2987Google Scholar). To determine its identity, we performed "in-gel" digestion with trypsin followed by separation on microbore high pressure liquid chromatography and N-terminal amino acid sequence of a tryptic fragment. The sequence NVPLPEF matched positions 282–288 of rat IDE (20Baumeister H. Müller D. Rehbein M. Richter D. FEBS Lett. 1993; 317: 250-254Google Scholar), confirming the identity of our recombinant protease and suggesting that 9B12 may indeed recognize a posttranslationally modified epitope in rat IDE that is not present in IDE expressed in bacteria. To characterize the activity of recombinant rIDE, we analyzed the degradation of [125I]insulin. After 1 h of incubation, rIDE was able to digest [125I]insulin and this activity was totally inhibited by EDTA-1,10-phenantroline and blocked to 80% in the presence of 1 μm unlabeled insulin (Fig. 1B). These results are fully consistent with the reported characterization of human recombinant IDE expressed in E. coli (34Chesneau V. Rosner M.R. Protein Expression Purif. 2000; 19: 91-98Google Scholar) and rat recombinant IDE produced in a baculovirus-insect cell system (32Mukherjee A. Song E. Kihiko-Ehmann M. Goodman J.P. St. Pyrek J. Estus E. Hersh L.B. J. Neurosci. 2000; 20: 8745-8749Google Scholar).Degradation of Aβ WT and Aβ Genetic Variants by rIDE—We followed the degradation of Aβ WT and Aβ genetic variants by rIDE using SDS-PAGE and Western blot with monoclonal 6E10 that recognizes an epitope within positions 1–16 of Aβ (38Pirttila T. Kim K.S. Mehta P.D. Frey H. Wisniewski H.M. J. Neurol. Sci. 1994; 127: 90-95Google Scholar) The relative amount of Aβ monomers and dimers that remained intact after incubation with rIDE was estimated by densitometry. This method allowed an accurate discrimination between monomeric and oligomeric Aβ as opposed to high pressure liquid chromatography or trichloroacetic acid precipitation (28McDermott J.R. Gibson A.M. Neurochem. Res. 1997; 22: 49-56Google Scholar, 29Perez A. Morelli L. Cresto J.C. Castaño E.M. Neurochem. Res. 2000; 25: 247-255Google Scholar). The absence of Aβ degradation products on Western blots may reflect the loss of the 6E10 epitope attributed to rIDE activity. Alternatively, the Aβ N-terminal fragments generated by the protease may be too small to be detected on SDS-PAGE. Under the conditions of our assay, the quantitation of degradation showed linearity up to 90 min of incubation (data not shown); therefore, a 60-min incubation was the time selected for the comparative analysis. As indicated in Fig. 2, A and B, monomeric Aβ WT, Aβ A21G, Aβ E22K, and Aβ D23N were degraded at 75.2 ± 3, 81.5 ± 9.2, 79.5 ± 12, and 84.7 ± 7.6%, respectively. rIDE was substantially less efficient in degrading Aβ E22Q and Aβ E22G monomers (42.3 ± 6 and 34.6 ± 6%, respectively, p < 0.01 as compared with Aβ WT). In the presence of 1,10-phenantroline, degradation of Aβ WT was inhibited almost completely (Fig. 2B). In contrast to Aβ monomers, degradation of dimers was consistently <10% for all of the variants studied (Fig. 2B), in agreement with the results reported recently on endogenous Aβ WT generated by Aβ PP-transfected cell lines (33Walsh D.M. Klyubin I. Fadeeva J.V. Cullen W.K. Anwyl R. Wolfe M.S. Rowan M.J. Selkoe D. Nature. 2002; 416: 535-539Google Scholar).Fig. 2Degradation of Aβ variants by rIDE.Panel A, Western blot analysis with monoclonal 6E10 of degradation of Aβ WT and Aβ genetic variants by rIDE. m, Aβ monomers; d, Aβ dimers. Panel B, densitometric quantitation of the immunoreactivity of Aβ as in panel A. m, monomers; d, dimers. Bars represent the mean ± S.E. of three independent experiments. *, p < 0.01 (Student's t test) as compared with Aβ WT.View Large Image Figure ViewerDownload (PPT)Characterization of Aβ Proteolytic Products—Aβ WT proteolytic products generated by rIDE were consistent with previous reports with the major sites of cleavage at His13-His14, His14-Gln15, Val18-Phe19, Phe19-Phe20, Phe20-Ala21, and Lys28-Gly29 (28McDermott J.R. Gibson A.M. Neurochem. Res. 1997; 22: 49-56Google Scholar, 32Mukherjee A. Song E. Kihiko-Ehmann M. Goodman J.P. St. Pyrek J. Estus E. Hersh L.B. J. Neurosci. 2000; 20: 8745-8749Google Scholar, 39Chesneau V. Vekrellis K. Rosner M.R. Selkoe D.J. Biochem. J. 2000; 351: 509-516Google Scholar). All of the Aβ fragments observed by MALDI-TOF MS are summarized in Table I, and Fig. 3 shows an schematic representation of the cleavage sites. Several fragments starting at Phe4 and Arg5 were found in Aβ WT and in all of the Aβ variants. These peptides were not the products of truncated synthesis as shown by MS analysis of undigested Aβ peptides and were not found when Aβs were incubated with rIDE in the presence of 1,10-phenantroline (data not shown). Moreover, Phe and Arg as P1′ residues are compatible with the known specificity of IDE (28McDermott J.R. Gibson A.M. Neurochem. Res. 1997; 22: 49-56Google Scholar, 41Pallitto M.M. Murphy R.M. Biophys. J. 2001; 81: 1805-1822Google Scholar). Therefore, it seemed probable that rIDE was capable of hydrolyzing Aβ Glu3-Phe4 and Phe4-Arg5 peptide bonds. In Aβ A21G, there was a consistent absence of fragments ending at Phe20 or starting at Gly21, suggesting the specific loss of a cleavage site. Notably, when we analyzed the digestion of rodent Aβ that present Gly instead of Arg at position 5, no fragments indicative of hydrolysis at the Phe4-Gly5 site were found, neither in rodent Aβ-(1–40) (Table I and Fig. 3) nor in rodent Aβ-(1–42) (data not shown), whereas all of the other cleavage sites present in human Aβ WT were conserved. The Dutch variant Aβ E22Q showed the apparent loss of sites at Val18-Phe19 and Lys28-Gly29, whereas in the Italian variant Aβ E22K, the latter cleavage site was also absent. This peptide bond has been reported to be resistant to IDE in Aβ WT-(1–42) (32Mukherjee A. Song E. Kihiko-Ehmann M. Goodman J.P. St. Pyrek J. Estus E. Hersh L.B. J. Neurosci. 2000; 20: 8745-8749Google Scholar), and therefore, its hydrolysis by IDE may depend upon oligomerization rather than primary structure. Alternatively, the presence of Gly at P1′ may impose a subsite restriction as suggested by the loss of cleavage sites in the Aβ Flemish and rodent variants. The Iowa type Aβ D23N differed notably from all of the other Aβ peptides studied. Fragments consistent with positions 1–26, 15–26, and 27–40 were found, indicating the cleavage at the Ser26-Asn27 bond (Table I and Fig. 3) and pointing to the possible importance of the Asp23 in the folding of the Aβ peptide.Table IMass spectrometry analysis of the cleavage products of Aβ variants by insulin-degrading enzymeAβ fragmentMolecular mass of Aβ variantsAβWTAβA21GAβE22KAβE22QAβE22GAβD23NAβrodentObs.Calc.Obs.Calc.Obs.Calc.Obs.Calc.Obs.Calc.Obs.Calc.Obs.Calc.Da1-131561.81561.6NF1562.61561.61561.91561.31562.21561.61562.11561.61466.31465.51-1416991698.31699.51693.71699.91698.71699.51698.71699.41698.71699.31698.71603.71602.61-182167.92167.3NF2165.62167.3NF2168.92167.32168.12167.32072.92071.21-192315.12314.52315.62314.5NFNF2315.72314.52315.32314.52220.22218.41-202462.62461.7NF2462.92461.7NFNFNFNF1-26NFNFNFNFNF3019.23019.2NF1-28NFNFNFNFNF3261.33261.53168.23166.54-131246.41246.31246.81246.312471246.31246.71246.31246.71246.31246.71246.31151.21150.24-141383.71383.513841383.51384.21385.513841383.413841383.51383.91385.5NF4-1920001999.2NFNFNFNFNFNF5-141236.31236.31236.81236.312371236.31236.61236.31236.61236.31236.61236.3NF14-20NFNFNF918.9918.1NFNFNF14-281719.41718.91705.81704.9NFNF1647.41646.9NFNF15-26NFNFNFNFNF1338.61338.5NF15-2815821581.81568.31567.8NFNF1510.51509.7NFNF15-40NFNF2650.52648.2NF2578.52579.1NFNF19-28NF1100.21099.2NFNFNFNFNFNF19-402180.42180.6NFNFNFNFNFNF20-40NF2020.32019.42033.52032.42033.82032.41962.11961.32033.12032.32034.62033.521-40NFNF1886.61885.31886.51885.21814.61814.11885.71885.21887.51886.227-40NFNFNFNFNF1327.71327.7NF29-40NF1084.91085.4NFNF1085.51086NFNF Open table in a new tab Fig. 3Schematic representation of cleavage sites within Aβ WT, Aβ rodent, and Aβ human genetic variants by rIDE.Arrows show the peptide bonds hydrolyzed by rIDE. Open arrowheads (▿) indicate loss of cleavage under the experimental conditions tested. The lines represent identity of amino acid sequence and cleavage sites. Amino acid substitutions in each Aβ variant are shown in circles.View Large Image