Structural Alterations in the Peptide Backbone of &Amyloid Core ...

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Kalamazoo, Michigan 49001, the TTDepartments of Pathology and Neurology, Oregon Health ... School of Medicine, 540 East Canfield Ave., Detroit, MI 48201.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 01993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 5, Issue of February 15, pp. 3072-3083,1993 Printed in U.S.A.

Structural Alterationsin the Peptide Backbone of &AmyloidCore Protein May Account for Its Deposition andStability in Alzheimer’s Disease* (Received for publication, September 9, 1992)

Alex E. RoherSO, Jonathan D. LowensonllII, Steven Clarken, CathyWolkow**, Rong Wang**, Robert J. Cotter**, Ilene M. Reardon$$, HeidiA. Ziircher-Neely$$$#, RobertL. HeinriksonSS, Melvyn J. BallTIV, and BarryD. Greenberg111) From the $Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, the TDepartment of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90024, the **Middle Atlantic Mass Spectrometry Laboratory, Department of Pharmacology and Molecular Sciences,The Johns Hopkins University Schoolof Medicine, Baltimore, Maryland 21205, $$Biochemistry and 11 IlCNS Research, Upjohn Laboratories, Kalamazoo, Michigan 49001, the TTDepartments of Pathology and Neurology, Oregon Health Sciences Uniuersity, Portland, Oregon 97201

The structureof &amyloid (@A)from Alzheimer disA major pathological characteristic of Alzheimer’s disease ease brains was examined to determineif post-trans- (AD)’ is the presence of amyloid-bearing senile plaqueswithin lational modifications might be linked to the abnormal the brains of afflicted individuals. PA core protein, the prideposition of this peptide in the diseased tissue.The BA mary proteinaceous component of AD amyloid deposits, has peptides wereisolated fromthe compact amyloidcores been characterized as a 39- to 43-residue peptide that accuof neuritic plaques and separated from minor glyco- mulates within theneuropil of vulnerable gray matter of the protein componentsbysize-exclusion high-pressure brain parenchyma and within the cerebromicrovasculature liquid chromatography (HPLC). This parenchymalBA (1-4). This core protein has been the target of intense study has a maximal length of 42 residues, but shorter forms since its initialisolation and partial characterization in 1984 with “ragged” NH2 termini are also present. Tryptic peptide analysis revealed heterogeneity in the fiA1-6 ( 5 ) . Sequence analysis of vascular PA reveals predominantly and PA6”‘ peptides, eachof which elutedas four peaks an NHz-terminalAsp (2, 5 ) . Similar analysesof parenchymal on reverse phase HPLC. Amino acid composition and PA have revealed a greater complexity, consistent with the presence of blocked (4, 6) and “ragged” NH, termini (1,7, 8). sequence analyses, mass spectrometry, enzymatic The PA core protein originates as an internal domain within methylation,andstereoisomerdeterminations rethe PA proteinprecursors(PAPPs),all of which arise by vealed that these multiple peptide forms resulted from structural rearrangementsof the aspartyl residues at alternative splicing of a primary transcript encodedbya @Apositions 1 and 7.The L-isoaspartyl form predom- unique gene on chromosome 21 (see Ref. 9 for arecent review). normally synthesized, secreted,and efficiently inates ateach of these positions, whereas theD-isoas- The PAPPs are partyl, L-aspartyl, and D-aspartyl forms are present in degraded in numerous mammalian tissues. Two processing lesser amounts. PA purified from the leptomeningeal pathways have been recently described, one secretory (10-13) microvasculature contains the same structural altera- and one endocytic (14-16). The secretory pathway results in tions as parenchymal @A,but is 2 residues shorter at an internal cleavage within the PA domain, precluding PA its COOH terminus. Using two different purification formation. Theendocytic pathway results in the formation of protocols, and using a synthetic flA”42 peptide as a potentially amyloidogenic PAPP fragments containing intact control, we showthat thesemodifications arose endog- PA domain; however, these fragments appear to accumulate enously and werenot caused by the experimentalma- at equivalent levels in both normal and AD brain (14, 17). nipulations. The abundanceof structurally alteredas- Hence, there is still noevidence for altered catabolism of PA partyl residues may profoundly affect the conforma- peptide inAD. Whether the accumulation of PA in AD results tion of the @A protein within plaque cores and thus from changesinthe processing pathway or from subtle significantlyimpactnormalcatabolicprocessesdechanges in thePA itself remains to be elucidated. signed to limit its deposition. These alterations may As a step toward clarifying amyloidogenic processes in AD, of therefore contribute to the production and stability we have undertaken a series of studies to identify and char@-amyloiddeposits in Alzheimer brain tissue. acterize in detail the components of amyloid cores purified from AD brain tissue. We show here that parenchymal and * This work was supported in part by National Institutes of Health vascular PA preparations contain a mixture of aspartyl isoGrant P30-AGO8017 (to M. J. B.), National Science Foundation forms at positions 1and 7. We furthershow that theseisomers Grants DIR-90-16567(to R. J. C.), and National Science Foundation DMB-89-04170and United States Public Health Service Grant GM- are formed by endogenous mechanisms and not asa result of modifications are 26020 (both to S. C.). The costs of publication of this article were theexperimentalmanipulations.Such defrayed in part by the payment of page charges. This article must known to alter the structure and function of avariety of

therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § T o whom reprint requests and correspondence should be addressed: Dept. of Anatomy and Cell Biology,Wayne State University School of Medicine, 540 East Canfield Ave., Detroit, MI 48201. Tel.: 313-577-1003. 11 Partially supported by a postdoctoral fellowship from the American Heart Association, Greater Los Angeles Affiliate. §§ Current address: Eli Lilly Pharmaceuticals, Indianapolis, IN.

The abbreviations used are: AD, Alzheimer’s disease; AdoMet, Sadenosyl-L-methionine; PA, P-amyloid; @Ad,P-amyloid dimer; PAm, P-amyloid monomer; PAPP, P-amyloid precursor protein; GdnHC1, guanidinium hydrochloride; GdnSCN, guanidinium thiocyanate; HPLC, high-pressure liquid chromatography; LDMS, laser desorption mass spectrometry; MIRL, membrane inhibitor of reactive lysis; PDMS, plasma desorption mass spectrometry.

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in Alzheimer 0-Amyloid

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10 min at 20 "C. The PA protein was then solubilized with either formic acid or GdnSCN. In the formercase, core preparations were dissolved in 80% glassdistilled formic acid at 4 "C. Following incubation for 20 min at room temperature, the solubilized cores were centrifuged in polyallomer tubes a t 435,000 X g for 10 min at 4 "C in a Beckman 100 TLA rotor to remove remaining insoluble particulates. The clear supernatant (3 ml) was dialyzed at 4 "C against 6 M GdnHCI, 0.1 M Tris-HCI, pH 8.5 using a 1,000-dalton cutoff dialysis tubing. This resulted in the formation of a white flocculent precipitate which was recovered by EXPERIMENTALPROCEDURES centrifugation a t 1,500 X g for 10 min a t room temperature. These pellets were redissolved in 80% formic acid and submitted to sizeMaterials-Collagenase CLS3 and DNase I (type D) werefrom Worthington. Guanidinium thiocyanate (GdnSCN), guanidinium hy- exclusion HPLC on a 300 X IO-mm Superose 12 column developed with the same solution at a flow rate of 0.2 ml/min with monitoring drochloride (GdnHCI), and formic acid were obtained from Fluka. Formic acid was glass-distilled in our laboratory prior touse. HPLC at 280 nm. Fractions containing PA peptide were concentrated by vacuum centrifugationand dialyzed against 8% aqueous betaine, columns included Superose 12 and C,, reverse phase(Pharmacia LKB Biotechnology Inc.), reverse phase (Cs-C,) Zorbax-protein plus followed by two changes of 2% betaine in0.1 M ammonium bicarbon(Du Pont),reverse phase C, (Vydac), Novapack CIR,and Resolve CIS ate, pH 7.8. In the lattercase, corepreparations were dissolved in 6 M GdnSCN (Waters). S-Adenosyl-~-[methyl-"C]methionine (["CIAdoMet, 52 a t room temperaturewithcontinuousstirringandthen mCi/mmol) was from ICN. Synthetic peptidesincluded a 42-residue for6h centrifuged in polyallomer tubes at 435,000 X g for 15 min at 4 "C in [jA (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGV a Beckman 100 TLA rotor to remove remaining insoluble particulates. V I A ) , a pentapeptide corresponding to the PA NH, terminus, The soluble PA was purified by fast protein liquid chromatography heginning with an L-isoaspartyl residue (L-iso-Asp-AEFR), and an octapeptide corresponding to PA residues 35-43 (VGGVVIAT). The usinga Superose 12column equilibrated and developed with6 M peptides were produced on an Applied Biosystems 430A synthesizer. GdnHCl in 6% acetic acid at a flow rate of 0.2 ml/min. Fractions They were characterized by amino acid composition, sequence analy- containing PA peptide were dialyzed against 0.2% betaine in 20 mM ammonium bicarbonate, pH 7.8, concentrated by vacuum centrifusis, and mass spectrometry. Human Tissue-Brains were obtained from 24 AD patients within gation and then dialyzed against 2% betaine in 0.1 M ammonium 4-12 h post-mortem. Left hemisphereswere submitted for histopath- bicarbonate, pH 7.8. Cerebrovascular Amyloid Purification-@A was also prepared from ological examination,andtherighthemispheres were frozen a t -70 "C, awaiting neuropathological diagnoses (19). During the proc- leptomeningeal vessels, following previously published techniques (2, ess of morphometric quantitation of AD lesions, we were able to 5) with some modifications. Meninges were gently teased from the underlying cortex and immersed in D A B S buffer. Large vessels (21 select those brains containing a high proportion of mature neuritic plaques exhibiting central amyloid cores. Four of these brains were mm diameter) were dissected freeand discarded. The remaining tissue was minced and washed twice with D A B S buffer, three times with also laden with leptomeningeal vascular amyloid deposits. Parenchymal Amyloid Plaque Core Purification--Brains were com- 20 mM Tris-HCI, pH7.6,0.8% NaCl at 2 "C, and then centrifuged a t bined in groups of four to provide sufficient starting material for the 500 X g for 10 min. Pellets were recovered, frozen in liquid N,, and ensuing analyses. Gray matter was dissected from coronal sectionsa t finely pulverized by mortar and pestle. The tissue was homogenized, cooled to 0-4 "C, and sonicated in 50 mM Tris-HCI, pH 8.0, 2 mM -10 "C. The tissue was minced and homogenized in a blender at 4 "C CaCI, a t 40-watt power output in 10-s bursts (Sonic & Materials, for 5 s in 20 volumes of disaggregation buffer (DAB-S buffer: 10 mM Inc., Danbury, CT). The sonicatewas then centrifuged at 25,000 X g Tris-HCI, pH 7.5, 0.25 M sucrose, 2 mM EDTA, 200 pg/ml phenylfor 30 min at 4 "C. The brownish top part of the pellet was recovered methylsulfonyl fluoride, 0.5 pg/ml leupeptin, 0.7 pg/ml pepstatin, 50 and resuspended in the same buffer and then incubatedwith 0.2 mg/ gg/mlgentamicinsulfate,and 0.25 pg/ml amphotericin B). The ml collagenase CLS-3 for 14 h at 37 "C. Insoluble material was homogenate was filtered through a series of stainless steel meshes recovered by centrifugation a t 100,000 X g for 30 min at 4 "C and the (700, 350, 150, 75 and 45 pm), adjusted to 1.2 M sucrose, and centri- PA extracted with 6 M GdnHCI, 0.1 M Tris-HCI, pH 10.2, for 72 h at fuged a t 25,000 X g for 30 min in a Beckman Type 19 rotor at 4 "C. room temperature with continuous stirring. Remaining particulate T h e pellets were resuspended in 12 volumesof D A B S buffer, filtered material was removed by centrifugation at 435,000 X g for 15 min at through a 45-pmstainless steelmesh, adjusted toa finalconcentration 4 "C in a Beckman 100 TLA rotor. As with the parenchymalamyloid of 1.9 M sucrose, and centrifuged at 125,000 X g in a Beckman Type preparations, subsequent PA purification from these clear superna28 rotor for 30 min at 4 "C. The solid pellets, which formed at the tants was performed in one of two ways, involving treatment with tops of the tubes, were removed with a spatula, pooled, then resus- either formic acid or with the chaotropeGdnHC1. pended and washed four times with 50 mM Tris-HCI, p H 8.0, 2 mM Some samples were applied to a 300 X 10-mm Superose 12 column CaCI,. Collagenase CLS3 (100 mg) and DNase I (5 mg) were added equilibrated with 80% formic acidand developed in this same solution. to the suspension (approximately 500 ml) followed by incubation a t A second chromatographic step was then performed on a 250 X 4.637 "C for 14 h with continuous shaking. The suspension was then mm reverse phase (C,-C,) Zorbax-protein plus column developed with centrifuged at 6,000 X g for 30 min a t 4 "C, the pellets washed three a linear gradient of 25-40% acetonitrile, 0.1% trifluoroacetic acid at times with 50 mM Tris-HCI pH 8.0, and solid SDS was added to a 0.8 ml/min, with monitoring a t 214 nm. @Afractions were dialyzed final concentration of 5%. After incubation at room temperature for against 2% betaine, 0.1 M ammonium bicarbonate, pH 7.8, as de2 h, the insoluhle material was recovered by centrifugation at 15,000 scribed above. X g and resuspended in 800 ml of the same buffer. Solid sucrose was Other preparations were applied in 500-pl aliquots to a 300 x 10added to a final concentration of 1.3 M, and the sample was centri- mm Superose 12 column equilibrated with 6 M GdnHC1, 6% acetic fuged at 150,000 X g for 45 min at 20 "C, resulting in the formation acid and developed with the same solution. Fractions containing PA of pellets above and below an intermediate supernatant. The top were pooled and dialyzed against 0.1% trifluoroacetic acid. Volume pellets and supernatantswere discarded, and the bottom pelletswere was reduced by vacuum centrifugation, and acetonitrilewas added to resuspended in 18 ml of Tris-SDS buffer (50 mM Tris-HCI, pH8, 1% a final concentration of 25%. SDS). This material was submitted to discontinuoussucrose density Production, Purification, and Initial Characterization of PA Fraggradient centrifugation by layering aliquots overa ladder of 2-ml ments-Tryptic digestion of the parenchymal and vascular DA pepsteps containing 2.2, 2.1, 2.0, 1.7, 1.4, and 1.3 M sucrose, respectively, tides, or of the synthetic PA peptide, was carried out at a mass ratio and then centrifuged at 200,000 X g for 1 h a t 20 "C in a Beckman of approximately 1:50 enzyme:substrate for 14 h at 37 "C. The reac41Ti rotor. The material recovered from the 1.4/1.7 M interface was tion was terminated by freeze-drying. The resulting peptides were layered over a second ladderconsisting of 2.3-ml steps containing2.2, separated by reverse phase HPLC using a 250 X 4.6-mm C,, column 1.8, 1.7, 1.6, and 1.5 M sucrose and then centrifuged under the Same with a linear gradient of 0-20% acetonitrile, 0.1% trifluoroacetic acid conditions. The amyloidcores,highly enriched at the 1.5/1.6 M developed over 90 min, followed by 20-60% acetonitrile, 0.1% trifluinterface, were washed with Tris-SDS buffer and centrifuged in 1.1 oroacetic acidfor 30 min a t a flow rate of 0.7 ml/min, with monitoring M sucrose at 300 X g for 15 minin conical 1-ml vials. The supernatants at 214 nm. Cyanogenbromide (CNBr) cleavage of the insoluble and top portionsof the pellets were discarded, and the bottom parts COOH-terminal tryptic peptide was performed in 80% formic acid of the pellets, containing the amyloid cores, were washed once with for 16h at room temperature. The resulting fragments were separated distilled water andtwice with 0.1% SDS and pelleted at 1,500 x g for under the same conditions as the soluble PA tryptic peptides.

polypeptides (18).The existence of these PA isoforms within AD amyloid cores may therefore affect the catabolismof these deposits with pathological consequences. Finally, we present evidence that the vascular PA is 2 residues shorter than the parenchymal PA at the COOH terminus, and show that this is moresoluble thanthecorrespondingparenvascular chymal

Structurally Altered Asp Residuesin Alzheimer @-Amyloid

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0 03

0 Oi

0 0'

c 0 03

b DAB

0 02

0 01

E o 0

aD e 4

flAm

Tryptic peptides were hydrolyzed in 6 M HCI, 1% phenol, for 2472 h at 108 "C in melting point tubes or in a PicoTag Work Station (Waters Instruments) at150 "C for 75 min. Amino acid compositions were determined in a n automated analyzer (Beckman 6300) by the ninhydrin reaction or in the form of phenylthiocarbamyl derivatives using a Novapack C1, column. Amino acid sequences were determined by Edman degradation on a model 470 AB1 gas-phase Sequencer with an on-line model 120 HPLC system and a Nelson chromatographic data system. The massof tryptic and CNBr peptideswas determined by plasma desorptionmassspectrometry(PDMS)andmatrix-assisted laser desorptionmassspectrometry(LDMS).PDMS (20, 21) was performed on a Bio-Ion Nordic(Uppsala, Sweden)model BIN-1OK timeof-flight mass spectrometerequipped with al-&i 25ZCfsource. LDMS (22) was performed on a time-of-flight instrument constructed at the Middle Atlantic Mass Spectrometry Laboratory as described in detail previously (23). Mass spectra of parenchymal and vascular PA peptides were calibrated using the caffeic acid matrix peak at m/z 181.1, acid peaks at m/z 190.2 and m/z the (Y-cyano-4-hydroxycinnamic 212.2, the protonated molecular ion peak for the tryptic PA fragment consisting of residues 6-16 at m/z 1337.4, or the protonatedmolecular ion for somatostatin at m/z 1638.8. Enzymatic Methylation of Altered Aspartyl Residues-The enzyme L-isoaspartate (D-aspartate) 0-methyltransferase (EC 2.1.1.77) was purified from the cytosol of human erythrocytes to a specific activity of about 5,000 units/mg of protein (1 unit = 1 pmol methyl groups transferred per min to an ovalbumin substrate a t 37 "C) as described (24). Thisenzyme catalyzes the methylation of the a-carboxyl group of L-isoaspartylresidues andthe8-carboxyl group of D-aSpartyl residues, but does not recognize L-aspartyl or D-isoaspartyl residues (18,25-28). It is therefore useful as an analytical probe for alterations in the peptide backbone at Asp residues. Intact SA (150-pmol aliquots) or its tryptic fragments (10-pmol aliquots) were incubated with 3.2 units methyltransferase at 37 "C for various times, in 20 p1 0.1 M sodium citrate, pH 6, containing10 pM ['4C]AdoMet. Reactions were terminated by addition of 50 pl of 0.2 M NaOH, 1% SDS, and methylation was quantitated by a vapor diffusion assay as described (29, 30). Assays were runinduplicate withnegative controls(no substrate) subtracted asbackground a t each time point. Stereoconfiguration Analyses of Altered Aspartyl Residues-Intact [jA or tryptic fragments containing the Asp residues at positions 1 and 7 were dried down in 6 X 50-mm glass tubes which had been preheated to 240 "C for 24 h to destroy any contaminating amino acids. Peptides were hydrolyzed with vaporized HC1 in uacuo using a PicoTag Work Station (Waters). Aspartic acid and serine residues were derivatized with N-acetyl-L-cysteine and orthophthalaldehyde (31), and the resulting diastereomers were separated isocratically on a 3.9 X 150-mm reverse phase Resolve C, column developed in a mixture of 95% Buffer A (50 mM sodium acetate pH 5.4), 5% Buffer B (80% methanol, 20% Buffer A) (32). Under these conditions, the fluorescent derivatives containing D-aSpartate and L-aspartateeluted a t about 6.3 and7.3 min, respectively. The derivatized D- and L-seryl residues eluted a t about 12 and 11 min, respectively. The fluorescence color constants for thesederivatives were determined with D- and Laspartate and D- and L-serine standards. Other amino acids present in the hydrolysates were quantitated by HPLC following derivatization with orthophthalaldehyde and 2-mercaptoethanol as described (30). To minimize experimentally induced artifacts, acid hydrolysis was performed for only 3 h a t 108 "C, conditions which are sufficient to release most of the Asp residues from peptide bonds (32). The extent of aspartate recovered from these 3-h hydrolysates was in all cases 90-100% of that recovered from 22 h hydrolysates performed a t this same temperature (not shown). RESULTS

Purification of Parenchymal and Vascular PA-Size exclusion HPLC of the parenchymal amyloid cores solubilized in

FIG. 1. Chromatographic profiles of parenchymal and leptomeningeal &amyloid. a-d show the purification of PA monomers (/,'Am) and dimers (flAd) using Superose 12 size-exclusion chromatography.The molecular masses of the indicated peaks were

determined from elution positionsof standard proteins. a, parenchymal PA proteins obtained from the filamentous cores found at the center of the neuriticplaques in AD brain. Cores were solubilized and the column was eluted in 80% formic acid; b, synthetic flA'-4' solubilized and eluted as in c, a;same source of material asin a. Solubilization was achieved in 6 M GdnSCN, and the column was eluted with 6 M GdnHC1; d, @Aobtained from the leptomeningeal vasculature of AD brain. &Amyloid was solubilized, and the column was eluted with 6 M GdnHC1. e, reverse phase C4 HPLC elution of vascular BAm obtained from material ind. The @Awas released at 36% acetonitrile concentration.

in Alzheimer 0-Amyloid

Structurally Altered Residues Asp J

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70

80

90

J

60

70

10

60

10

10

90

YN

0 li

0 01

0 01

0

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formic acidyielded six fractions with estimated massof >200, 200, 45, 15, 10, and 5 kDa (Fig. la). In a related study, we showed that the first three fractions contain an assortment of glycoproteinsincludingrul-antichymotrypsin and PAPP, whereas the fractions at 15, 10, and 5 kDa contain trimeric, dimeric, and monomeric forms of PA, respectively.' These last three fractions contain virtuallyall the PA present in the parenchymaltissue. Althoughinsoluble material was discarded during the purification procedure, this material consisted primarilyof lipofuscin granules, which yielded a different amino acid composition than the soluble PA and did not react with antibodies raised against PA (33). As a control, synthetic was submittedtothesame formic acid solubilization andchromatographic proceduresapplied tothe parenchymal cores. This material co-eluted with the peaks identified as monomeric and dimeric forms of the parenchymal PA (Fig. Ib). When parenchymal cores were solubilized was in 6 M GdnSCN instead of formic acid, a broad peak obtained followed by 10- and 5-kDa peptides corresponding to these dimeric and monomeric PA forms (Fig. IC).Leptomeningeal vascular amyloid yielded essentially the same elution pattern by size-exclusion chromatography when solubilized in either formic acid or in GdnHCl (Fig. Id). Rechromatography of these PA-containing fractions on C4 reverse phase yielded purified cerebrovascular PA (Fig. le). All subsequent analyses were performed on PA monomers purified from boththeparenchymaand cerebrovasculature, corresponding to the 5-kDasize-exclusion peak. Post-translational Modifications in Parenchymal PA Detected by Tryptic Dcgestion-In ordertosearch for posttranslational modifications,vascular andparenchymal PA monomers solubilized in either formic acid or guanidinium salts were hydrolyzed with trypsin, and theresulting peptides were fractionated by reverse phase HPLC. This procedure was expected to yield four fragments, designated T p l DAEFR), Tp2 (PAfi-lfi, HDSGYEVHHQK), Tp3 LVFFAEDVGSNK), and Tp4 (pA29-4', GAIIGLMVGGVVIA). Although this result was indeed obtained with formic acid-treatedsynthetic PA1-42(Fig. 2c),tryptic hydrolysates of parenchymal and vascular preparations gave a much more complex pattern of peaks. The amino acid compositions of peaks A-C in the parenchymal PA digest(Fig. 2a) were essentiallyidentical,each consisting of the residues expected for T p l (PA"', Table I). However, only peak C exhibited the expected sequence by automated Edman degradation.Sequence analysis of peak A revealed only PA residues 2-5 (AEFR), suggesting that this fraction contained a mixture of the two peptides AEFR and DAEFR, the latter being resistanttoEdman degradation.

90

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mln

FIG. 2. HPLC analysis of tryptic and CNBr peptides of parenchymal and meningeal j3A. Tryptic peptides (a-d) and CNBr peptides ( e ) were separated by Clx reverse phaseHPLC usinga shallow gradient as defined under "Experimental Procedures." PA-

A. E. Roher, K. C. Palmer, E. C. Yurewicz, M. J . Ball, and B. D. Greenberg, submitted for publication. ______ derived peaks are identified by the capital letters A-N. Analytical data presented throughout the text and tables correspond to this nomenclature. a, tryptic digest of parenchymal @Amsolubilized in formic acid (see Fig. l a ) . Trypsin was expected to yield four pA"' fragments designated Tpl (j3A"')), Tp2 (PAfi"'), Tp.3 and Tp4 (/3A29-42). T h e T p l peptide is found in peaks A-C, theTp2 peptides are found in peaks D-I, and Tp3is found in peak J . Tp4 was insoluble and recovered by centrifugation. b, tryptic digest of parenchymal @Amsolubilized in GdnSCN (see Fig. Ib). c, tryptic peptides derived from synthetic & 4 - 4 2 solubilized in formic acid(@Amonomers @ A m ) from Fig. IC). Synthetic j3A'-42 yielded only tryptic peptides 1-5 (C), 6-16 (I), and 17-28 ( J ) .COOH-terminal j3A2"4' was again insoluble and recovered by centrifugation. d, tryptic digest of leptomeningeal @Amfrom Fig. le. e, chromatogram of insoluble COOHterminal Tp4 (@A'"'') solubilized in formicacid and cleaved with CNBr.The chemicalhydrolysis at MetT5yielded CNBrpeptides PA1"42 ( L ) and pA2"Rs ( M and N ) in the forms of homoserine and homoserine lactone.

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TABLE I Expected and observed tryptic fragments of parenchymal PA The expected tryptic fragments are shown on the left-hand side of the table (Tpl, Tp2, Tp3, and Tp4). The eleven PA tryptic fragments actually resolved from AD brain tissue are listed on the right-hand side of the table, in the order of elution from the CISreverse phase HPLC column. The best assignments of their sequences are noted, as determined by a combination of amino acid composition and sequence analyses. Tryptic fragment

@Aresidues

TP 1

Peaksequence Predicted

DAEFR

quence

A AEFR

B TP2

TP3 TP4

PA6-

PA'7-28 PA2'-'

HDSGYEVHHQK

LVFFAEDVGSNK GAIIGLMVGGVVIA

TABLE I1 Molar ratios (%) of tryptic peptides (residues6-16) obtained from cortical and meningeal PA

60.7 48.3

C D E, F G H I J

@AComposition resi- and/or dues

se-

observed

DAEFR gA2-5 PA1-5 PA10-16 PA'*I6 &49-16

PA"'6 PA6-16 PAS16 PA'7-28 PA29"z

DAEFR YEVHHQK DAEFR YEVHHQK GYEVHHQK SGYEVHHQK HDSGYEVHHQK HDSGYEVHHQK HDSGYEVHHQK LVFFAEDVGSNK GAIIGLMVGGVVIA

of our solubilization procedure. We therefore substituted6 M GdnSCN for formic acid to solubilize the parenchymal cores and purified the PA by size-exclusion HPLC in GdnHC1. As Tp2 peak G Tp2 peak H Tp2 peak I seen in Fig. 2b and Table 11, the ratios of peptides G-I were similar regardless of the solubilization protocol. We also inCortical @A,HCOOHtreated vestigated whether formic acid might affect the structure of Lot 1" 17.6 21.7 thesynthetic peptide. Treatment with 80% formic acid Lot 2" 38.1 13.6 at room temperature for up to 24 h followed by dialysis and 19.9 26.4 Lot 3" 53.7 trypsin hydrolysis yielded the expected fragmentsalmost 23.6 21.7 54.7 Lot 40 quantitatively. As shown in Fig. 2c, peaks C (Tpl), I (Tp2), 22.6 19.7 57.7 Lot 5" and J (Tp3) predominated, withonly a very small amountof Mean f S.D. 55.0 f 4.16 18.5 f 2.78 26.5 ? 6.02 material elutingat theposition of peak B. Notably, there was Cortical PA, GdnSCNno observation of Tp2 peaks G and H. Sequence analysis treated confirmed the identitiesof these peptides. 25.155.7 19.2 Lot 1" T o demonstrate further the correspondence of the tryptic Meningeal PA, GdnHC1peptides from synthetic PA"** to those obtainedfrom parentreated chymal amyloid, aliquots of these two tryptic digests were Lot 1 6 5.40 14.3 80.3 mixed and co-chromatographed. The syntheticTpl and Tp2 a Each lot represents a pool of four cerebral hemispheres. 'Representsa pool of leptomeninges from four cerebral hemi- co-eluted with brain peptides C and I, respectively (data not shown). Taken together, these results show that the additional spheres. parenchymal PA tryptic peaks with the compositions of T p l and T p 2 were not induced by the formic acid solubilization Similarly, sequence analysis of peak B revealed no T p l se- procedure. It wasalsoclear thatanexplanation for the quence a t all. These resultswere not entirelyunexpected since presence of several chromatographically resolvable Tpl and i t has been reported that parenchymal amyloid cores contain T p 2 peptides could not be derived from conventional amino @A with ragged NH2 termini (7,8), and full-length@A"42 has acid compositional and sequence analyses. been reported to be resistant to the Edman degradation reResolvable Parenchymal PA Tryptic Fragments with Idenaction and perhaps NH2-terminally blocked (4, 6). tical Amino Acid Compositions Also Exhibit Identical MolecuPeaks D-I (Fig. Sa) were all derived from Tp2. Composi- lar Mass-Mass spectral analyses (LDMS and PDMS) were tional analysis of peak D was consistent with the sequence of pursued in order to reveal any post-translational modifica(Table I). Composition and sequence analyses ofpeaks tions which might alter the molecular mass of the multiple E and F indicatedpeptidescorrespondingto PA8"6 and Tpl and Tp2peptides. The results,which are summarized in PAg-l6, which presumably resultfrom the ragged NH2 termini. Table 111, were essentially identical for preparations solubiPeaks G-I all contained peptides with the composition of lized in either formic acid or GdnSCN. the complete sequence expected for Tp2; however, peak LDMS molecular ion regions obtained for T p l samples AI was the onlyone which yielded the completesequence C are shown in Fig. 3, a, b, and c, respectively. The peaks at extending from His' to Lys" by Edman degradation. Sequenc- m/z 636.9, 637.4, and 638.1 correspond to theMH' for PA"5, ing of peaks G and H stopped after the NH2-terminal His (PA consistent with the calculated mass of 636.7. The peaks at ml residue 6), suggesting the presence of an alteration in the z 658.1, 658.0, and 659.7 correspond to the respective MNa+ peptide backbone a t position 7 which precluded further steps ions. The peak at m/z 522.0 in Fig. 3a corresponds to the in the Edmandegradation. This "Tp2 triplet"was a constant MH+ ionfor theNH2-terminallytruncated found prefeature in the PA tryptic digestions, with only minor differ- viously in peak A of the Tpl triplet by sequence analysis, and ences in the ratios of these peaks observed in extracts from consistent with the calculated mass of 521.6. PDMS peaks six independent preparations of cerebral hemispheres (Table were in close correspondence to these values (Table 111). In 11). addition, PDMS peaks corresponding to the (M + 2Na-H)+ Since PA has been reported to be modified structurally by ion of PA1-' were observed in sample A. Thus, the mass of Tpl peaks A-C appeared to be identical within experimental exposureto formicacid(34, 35), we testedwhetherthis resolution of Tp2 into peaksG, H, and I might be an artifact error.

3077

Structurally Altered Asp Residues in Alzheimer fi-Amyloid TABLE111 Matrix-assisted LDMS and PDMS ofpeptides deriued from the DarenchymaE B-am.~loid Peak LDMS" PDMS"

C D E

F G H

I J K L M N

Mass (calculated)

636.7 DAEFR 521.6 (1) 522.0 523.3 AEFR 1199.4 (1) 1197.8'1200.9' DSGYEVHHQK 10-16 940.1 (1) ND 942.0 YEVHHQK 1-5 636.7 (1) 637.4b 638.6 DAEFR 521.6 (1) ND 523.7 AEFR 1199.4 (1) 1223.2d1201.0 DSGYEVHHQK 10-16 940.1 (1) 941.4' 941.5' YEVHHQK 1-5 636.7 (1) 638.1b 638.0' DAEFR 1114.2 (1) 1113.4' 1115.0' FFAEDVGSNK 940.1 (1) 942.7 943.1' YEVHHQK 1084.3 (2) 1082.8' ND SGYEVHHQK 9-16 997.2 GYEVHHQK (2) 996.1' ND 1084.3 SGYEVHHQK (2) 1084.1' ND 9-16 997.2 GYEVHHQK (2) 997.3' ND 1336.5 (1) 1337.4"1337.6' HDSGYEVHHQK 1336.5 (2) ND 1337.7' HDSGYEVHHQK 1336.5 (1) 1337.9'1338.0 HDSGYEVHHQK (2) 1337.4'1337.8' HDSGYEVHHQK 6-16 1336.5 1336.5 (1) 1337.5'1337.9' HDSGYEVHHQK (2) 1337.2'1337.4' HDSGYEVHHQK 6-16 1336.5 1325.6 (3) ND 1325.0' LVFFAEDVGSNK 1325.6 (2) ND 1326.0' LVFFAEDVGSNK 1085.5 (3) ND 1085.0 GAIIGLMVGGVV 613.8 613.7' VGGVVIA (4) ND 625.8' 627.5' GAIIGLM (4) ND 625.8' (4) ND 627.5' GAIIGLM

A (1) 636.9'638.0b

B

Sequence

'* residues 1-5 2-5 7-16 2-5 7-16 19-28 10-16 8-16

1

8-16

637.4

6-16 6-16 6- 16 6-16 17-28 17-28 29-40 36-42 29-35 29-35

Protonated molecular ions unless otherwise indicated. Numbers in parentheses indicate: samples from tryptic digest of GdnSCNtreated parenchymal PA (l),samples from tryptic digest of formic acid-treated parenchymal PA (21, samples from tryptic digest, of leptomeningeal BA (3), and samples from CNBr digest of COOHterminal Tp4 peptide of parenchymal PA (4). ND, not determined. Also observed MNa+ and (M + 2Na - H)' ions. Also observed MNa' ions. MNa' ion. 'Also observed Mna+, (M + 2Na - H)' and (M + 3Na - 2H)' ions. Conversion of Met to homoserine lactone.

1241.4

50-

loa0

..

1500

aP 630.1 C.

'

LDMS of Tp2 samples D-F revealed m / z peaks consistent with the compositional and sequence analyses (Table 111). LDMS molecular ion regions obtained for Tp2 samples G-I are shown in Fig. 4, a, b, and c, respectively. The peaks at ml z 1337.4,1337.9, and 1337.5 correspond tothe MH' for I 1 PA6-l6, consistent with the calculated mass of1336.5. The lo00 1600 d S peaks at m / z 1359.6, 1360.9, and 1358.9 correspond to the FIG. 3. Matrix-assisted laser desorption mass spectra of the respective MNa+ ions. Minor peaks at m/z 1319.6 and 1320.7 in Fig. 3, a and b, most likely resulted from the loss of water isomeric Tpl samples. Material was GdnSCN-treatedparenchymal from the molecular ion. PDMS peaks were again in close PA obtained from the chromatogram in Fig. 2b. a, sample A; b, sample B; c, sample C. The peaks at m/z 636.9, 637.4, and 638.1 correspond correspondence to these values (Table 111). Thus, the mass of tothe MH' for consistent with the calculated mass of 636.7 Tp2 peaks G-I also appeared to be identical within experi- kDa. The peaks at m/z 658.1,658.0, and 659.7 correspond to the mental error. respective MNa' ions. These mass spectral analyses also revealed cross-contamination of the various peaks which was not detectable at the these BA tryptic fragments. It was concluded that thefeatures sensitivity of the compositional and sequence analyses. For of the T p l and Tp2 peptides accounting for their resolvable example, some spillover from T p l sample A was evident chromatographic characteristics must rely on structural modin the PDMS analysis of T p l sample B. T p l samples B and ifications which conserve their mass. C also bothcontained the peptide from Tp2 sample D, Structural Alterations Resulting in Conserved Molecular despite the fact that sequence analyses revealed this contam- Mass but Altered Chromatographic Behavior Might Be Due to inant only in peak B. Because of the sensitivity of these AspartylIsomerization-A review of the primarystrucmethods, it is not surprising that two additional peptideswere ture revealed that Asp was the intractable residue in the revealed which were not observed by amino acid sequencing Edman analyses of both T p l and Tp2, located at the NH, or composition analysis; T p l samples B and C contained a terminus of T p l (PA residue 1) and atposition 2 in Tp2 (PA Tp2 derivative PA7-16, and Tp2 sample D contained a Tp3 residue 7). As depicted in Fig. 5, aspartyl residues are known derivative /3A1g-28 (Table 111). to undergo nonenzymatic structural modifications (36-40). Hence, despite their sensitivity, these mass spectral anal- The attackby a peptide backbone nitrogen on the side chain yses also failed to explain the chromatographic behavior of carbonyl of an adjacent aspartyl residue can result in the

Structurally Altered Asp Residues in Alzheimer @-Amyloid

3078

explain the chromatographic and biochemical characteristics of these peptides. Identification of Altered Asp7 in Parenchymal PA-The presence of altered aspartyl residues was investigated using the enzyme L-isoaspartate (D-aspartate) 0-methyltransferase (EC 2.1.1.77) as an analytical probe. This enzyme exhibits a stereoconformational specificity, transferring methyl groups to peptide substrates containingL-iso-Asp or D - A s ~but , not L-ASP or D-iso-Asp residues (18, 27, 42), thus providing an analytical assay for these abnormal forms. These analyses revealed that parenchymal PA indeed contains isomerized aspartyl residues. As seen in Fig. 6a, purified intact parenchymal PA is a good methyl-accepting substrate for this enzyme, whereas the corresponding synthetic peptide, in which the Asp residues should be predominantly in the normal L - A s ~configuration, was a poor substrate. Tp2 peptidesG, H, and I were investi1337.9 gated similarly, in order to determine whetherPA position 7 b. was the site of enzyme-directed methylation. The trailing shoulder of peak I, here called I-bis, was also investigated. This shoulder is more evident in parenchymal PA extracted with GdnSCN (Fig. 26) than with formic acid (Fig. 2a) and not a t all evident in the tryptic digest of the synthetic PA1-42 (Fig. 2c) or in cerebrovascular PA (Fig. 2d). As indicated in Fig. 6b, sample G was the best acceptor, with almost 30% of the peptide molecules methylated after 6 h. Sample H was methylated about half as well, whereas neither I nor I-biswas significantly labeled. In conjunction with the block observed during Edman degradation, these results indicated that the predominant peptide in Tp2 sample G contained an L-isoAsp residue. The methylation observed in sampleH was likely due to cross-contamination between peaks G and H, which were less well resolved from each other thanfrom peak I (see Fig. 2, a and b). Racemization at PA Asp7 was investigated in peptides G, 1337.6 H, I, and I-bis, and in peptideIfrom the synthetic peptide, in order to distinguishL-ASPand L-iso-Asp from the corresponding D-stereoisomers. Following acid hydrolysis of these peptides, the PA hydrolysates were derivatized with N acetyl-L-cysteine and orthophthalaldehyde to generate fluorescent aspartyl diastereomers which could be resolved by reverse phase HPLC (31, 43). As shown in Fig. 7 and Table IV, 98% of the Asx residuesreleasedfrom thesynthetic were in the L-configuration. Thus, no more than 2% of the racemizedresiduesobservedin parenchymal PA were expected to have been created by the experimental manipulations, It was therefore significant that D-ASX accounted for 11% of the total Asx residues in purified parenchymal PA (Table IV). This difference between parenchymal and syn1200 d z 1400 thetic PA was further emphasizedin analyses of the Tp2 FIG. 4. Matrix-assisted laser desorption mass spectra of the isomeric Tp2 samples. Material was GdnSCN-treatedparenchymal samples G-I. These studies revealed that 91% of the Asx (lrA obtained from the chromatogram in Fig. 2b. a, sample G ; b, sample residues in sample G existed in the L-configuration, whereas H; c, sample I. The peaks at m/z 1337.4,1337.9,and 1337.5correspond 54% of the Asx residues released from sample H were in the t o the MH' for @A6-l6,consist,ent with the calculated mass of 1336.5 D-configuration (Fig.7a, Table IV). In comparisons of samples kDa. The peaks at m / z 1359.6, 1360.9, and 1358.9 correspond to the I and I-bis, the latterwas enriched in D-ASX (24%) compared respective MNa' ions. with sample I (12% D-Asx). For reasonsstated above, cross-contamination between formation of a five-membered succinimidering. This cyclized samples G and H likely accounted for the D-ASXin sample G structure is proneto racemization and further hydrolysis, and for the L - A s ~(in theform of L-iso-Asp) in sample H. The producing aspartyl and isoaspartyl residues in both the D- andfinding that approximately 50% of the iso-Asp in sample H r>-stereoconfigurations (18, 25, 26, 36). Notably, these struc- was in the L-configuration is consistent with the previous tural modifications do not alter the mass of the corresponding observation thatsample H was enzymatically methylated peptides. In the case of isoaspartyl formation, the peptide about 50% as well as sampleG (Fig. 6 ) . Peaks I and I-biswere backbone is transferred from the a-carboxyl to theside chain clearly unresolved (Fig. 2, a and b ) , similarly accounting for &carboxyl, resulting in a significant structural perturbation the apparent cross-contaminationbetween the corresponding of t,he peptide backbone, as well as resistance to the Edman samples in these analyses. When all the data are taken together(amino acidcompositions,sequenceanalyses,mass reaction (41). The possibilityfor structuralalterationsat stereoisomer these Asp residues was therefore explored in an attempt to spectral analyses, enzymatic methylation, and

1

3079

Structurally Altered Asp Residues in Alzheimer 6-Amyloid

FIG. 5. Spontaneous reactions leading to the formation of altered aspartyl residues in polypeptides. T h e reactions in this pathway have been well characterized using synthetic peptides in uitro. At pH 7.4 and 37 "C, Laspartyl residues can degrade to the Lsuccinimidylform with tl/? values as shortas 41 days for an Asp-Gly sequence,and168days for an Asp-Ser sequence(37).Insimilarpeptides, t1/2 values of 19.5 h for racemization of the 1,-succinimide and 2.3 h for hydrolysis of the succinimide to the normal and isoasparty1formshavebeenobserved (36). Although not shown in this figure, each of these reactions is reversible. The attachment of the residue to the peptide backbone is represented by the bold lines.

L -succinimidyl

e

D

e

D

0

0

L- isoaspartyl

D- isoaspartyl

studies), it is clear that the chromatographic differences observed among the Tp2 peaks G, H, I, and I-bis were due to the stateof the Asp residue at PA position 7, with G containing primarily L-iso-Asp, H containing D-iso-Asp, I containing LAsp, and I-bis containing D - A s ~ . ~ Identification of Altered Asp' in Parenchymal PA-As discussed above, Edman degradation revealed the expected primarystructureDAEFR) only insample C of theTpl "triplet," despite the fact that samples A-C all provided amino acid compositions andmassspectralresults expected for DAEFR.SincetheNH2terminus of/3A1-5 isanaspartyl residue, a situation similar to Asp7 in the Tp2 peptides was suspected. To test this possibility, theaspartylstereoconformation was examined in acid hydrolysates of samples A, B, C and "A-bis," the shoulder to the right of peak A in Fig. 2, a and b. As shown in Fig. 7b andTable IV, sample C contained primarily L-ASPin both parenchymal and meningeal PA samples solubilized with formic acid. Combined with the observationthatthepeptide insampleCcould be completely sequenced, this supports its designation as the native L-ASPcontaining peptide. Sample Aalso containedprimarily Laspartate, whereas sample A-bis contained primarilyD-aspartate. It is likely that these partially resolvable peaks contain the L- and D-iSOaSpartyl isomers, respectively, sinceboth were resistant to Edman degradation. Furthermore, sampleA was shown to co-elutewitha synthetic peptide containing the (not shown). The pressequence L-iso-Asp-Ala-Glu-Phe-Arg ence of D - A s ~and L-iso-Asp in these samples could not be confirmed enzymatically, however, since the L-isoaspartate (D-aspartate) methyltransferase does not effectively methylate NH2-terminalresidues (42, 44). Nevertheless, it remains likely thatthe peptidesin samples A, A-bis, B,and C differ exclusively by the conformation of the aspartylresidue at position 1, reminiscent of the situation confirmed at PA residue 7, containing primarily L-iso-Asp, D-iso-Asp, L - A s ~ , a n d D - A s ~respectively. , Racemization of Seryl Residues in PA-It was previously reported that PA from AD brain contains racemized Ser as well as racemized Asp residues (45). We therefore measured The lack of observable methylation of the D-Asp-containing peptide probably results from the lower affinity of the methyltransferase far this residue in comparison to L-isoaspartyl residues (27).

Q

o II

0

D-aspartyl

the D-Ser content in the various PA6-16 isoforms. In each of these peptides, 4-9% of the serine exists in the D-configuration (not shown). However, the presence of D-Ser did not appear to affect the chromatographic behavior of the PAfi-16 peptides. The COOH Termini of Parenchymal and Leptomeningeal PA Differ by Two Amino Acids-When purifiedfrom the parenchyma, full-length PA monomers were always retained on C4reverse phase columns, as was the synthetic PA"@', whereas leptomeningeal PA monomers eluted as a distinct peak (Fig. le). Analysis of the respective COOH-terminal tryptic fragments (Tp3 and Tp4) provided an explanationfor these differences in chromatographicbehavior. Composition and sequence analyses of peak J from the parenchymal preparations (Fig. 2) revealed the complete primary structure expected for Tp3. The COOH-terminal Tp4 peptide PA29-42 obtained from the parenchymal PA formed an insoluble precipitate. This peptide was recovered by centrifugation and resolubilized in 80% formic acid. Cleavage with CNBr was expectedto yield two fragments, GAIIGLM in which the Met had been modified to homoserine A CIS reand homoserine lactone, and VGGVVIA (pA"6-42). verse phase HPLC elution profile for this CNBr digest is shown in Fig. 2e. Composition and sequence analyses revealed that peak L contained the COOH-terminalPA"""2, and peaks M and N contained the two PA2g-3sforms, as expected. Tryptic digestion ofPA purified from the leptomeningeal microvasculaturerevealed major peptidescorresponding to Tpl (peakC), Tp2 (peak I),Tp3 (peakJ ) , and a novel peak K (Fig. 2d). Composition analysis revealed that peak K corresponded to PA29-40,which is twohydrophobicresidues shorter than the corresponding COOH-terminal tryptic peptide (Tp4) obtained from parenchymal cores. This COOHterminal truncation evidently resulted in sufficient solubility of the vascular PA for recovery by reverse phase HPLC. These results were confirmed by mass spectral analyses of these tryptic peptides and the CNBr subfragments of the insoluble parenchymal Tp4. PDMS for Tp3 (peak J in both parenchymal and meningeal samples) revealed a mass consistent with the calculated value of 1325.6 (Table 111). The CNBr subfragments of parenchymal Tp4 (peaksL, M , and N in Fig. 2e) also gave the expected mass. PDMS for peptide I, revealed a peak at m/z 613.7, consistent with the calculated mass of 613.8 for PA36-4L.Peptides M and N both yielded

Structurally Altered Asp Residues Alzheimer in@-Amyloid

3080 8~

7

1

0 Alzheimer OA

0

A Synthetic OA

6.

0

0

40

0 0

8

0

A

,'

0

100

. 0.30 u

-

0 .-

50.25a

-

%o.zo

--

. . A Tp 2 ( i ) a Tp 2 IIbis) a Tp 2 ( H I

.

.

.

.

h

A

A

200 Time ot Incubation (mint .

.

300

.

4

8

4

8 4 Tine Ininl

8

4

8

4

8

4

I

4

8

8

4

8

4

8

400

.

. 0

. O .

0 0

0

oTp2(Gl

0

L

0

u

n

0.15

-

0.10

-

3

e

0

'

0

Q

f

f 0.05-

8

,8

0 0

e

:

a

0

f ~

0

50

100

8

~~

150

~

a a -

4 . A

~~

200

250

300

350

Time of Incubation Imin)

FIG. 6. Detection of altered aspartyl residues in 014and j3A tryptic peptides by methylationwith L-isoaspartate (D-aspartate) methyltransferase. Polypeptides (150 pmol of parenchymal or synthetic SA"42 or 10 pmol tryptic peptide) were incubated with 3.2 units of the methyltransferase and 10 PM [I4C]AdoMet a t 37 "C for the times indicated and the number of methyl groups transferred to the peptide substrate quantitated asdescribed under "Experimental Procedures." Parallel incubations done in the absence of added peptide were subtracted from each time point. Top panel, parenchymal PA and synthetic /3A"". Bottom panel, parenchymal PA Tp2 samples G, H, I, and I-bis (see text).

peaks atm/z 627.5, corresponding to the protonated molecular ion MH+ of with the COOH-terminal Met converted t o homoserinelactone. PDMS for peptide K, the COOHterminal tryptic peptide of the meningeal PA, yielded a peak at m/z 1085.0, consistent with the expected value of 1085.5 calculated for the MH' ion of /3A29-40. Thus, PDMSconfirmed that theleptomeningeal PA terminates atresidue 40, whereas the parenchymal PA extends toresidue 42. These analyses do not eliminate the possibility that leptomeningeal PA may also contain some PA"", since any meningeal would havebeenremovedfrom the PA samples during the C, reverse phase chromatography step. It is clear, however, that parenchymalcores contain @A"" almost exclugenerated sively, since tryptic digestion of meningeal peptide K (see Fig. 2 4 , which was never seen in digests of the parenchymal material (Fig. 2, a and b ) . Thus, any would have to constituteonly an extremely minor fraction of parenchymal PA. Similar PA Structural Modifications Occur within the Leptomeningeal Vasculature-In meningeal @Apreparations, the most abundant NHz-terminal T p l and Tp2 peptides corresponded to the normalL - A s ~ stereoconformers (peaks C and I). Multipleisoforms of these peptides were nevertheless resolvable, although peaksA, B, G, and H were less prominent

r

4 line Inin)

FIG. 7. Stereoconformational determination of Asp'and Asp' in parenchymal 0A. Aspartyl residues were released from the indicatedpeptides by mild acidhydrolysis of formic acid-treated parenchymal PA. These residues were then derivatized and resolved by reverse phase HPLC asdetailed in the text.Top panel,analysis of Asp' (tryptic peptides in Tp2 samples G, H, I, and I-bis). Bottom panel, analysis of parenchymal Asp' (tryptic peptides inT p l samples A, A-bis, B, and C) and meningeal Asp' peptide C ( M - C ) .

TABLE IV T h e stereoconfiguration of the aspartyl residue atPA position 7 The top section of the table presents the data for the synthetic peptide and for parenchymal @A'-42extracted from AD brains. The remaining three sections refer to tryptic peptides derived from the parenchymal PA"' or vascular /3A"". Samples were hydrolyzed as described under "Experimental Procedures." Stereoconformation of the released aspartyl residues was determined by derivatization with N-acetyl-L-cysteine and orthophthalaldehyde. Separationof the resulting fluorescent diastereomers by reverse phase HPLC isshown in Fig. 7. Values obtained for an equivalent amount of 0.1% trifluoroacetic acid carried through the same steps were subtracted from the experimental figures as background blank.These values arethe average of duplicate determinations. Peptide

Mol % L - A s ~

Mol % D - A s ~

Synthetic PA1-42 Parenchymal

98 89

2.0 11

Tp2 Tp2 Tp2 Tp2

sample G" sample H" sample I" sample I-bis"

91 46 88 76

54 12 24

Tpl Tpl Tpl Tpl

sample Ab sample A-bisb sample Bb sample Cb

96 48 68 94

4.0 52 32

99

1.0

Tpl sample

c'

9.0

6.0

"Tryptic peptides containing residues 6-16 of parenchymal pA (Tp2 samples). Tryptic peptides containingresidues 1-5 of parenchymal SA (Tpl samples). Tryptic peptides containing residues 1-5 of meningeal SA.

Structurally Altered

Asp Residues in Alzheimer 0-Amyloid

3081

than in parenchymal samples (compare Fig. 2, a and dl. These vaF, potentially limiting succinimide formation due to its results were most likely due to endogenous aspartyl isomeri- bulkyhydrophobicside chain. It is alsopossible, however, zation within PA from both brain locations. The alternative that higher order structure or limited local flexibility may contribute to the stabilityof Aspz3. possibility that these minor peaks might have arisen from Due to earlier reports that PA peptides can by formylated contamination of meningeal PA with parenchymal cores is whensolubilized in formicacid (34, 35), we investigated less likely, again due to the C, reverse phase HPLC step employed in the meningeal PA purification (uide ante). If whether our purification protocol might have induced these peaks A, B, G, and H were actually contaminants, parenchy- structural modifications. We found noevidence for this. The same modificationswere observed whether AD amyloid cores mal PA would have to exist asa mixture of (3A1-40plus were solubilized in formic acid or in guanidinium salts. FurIt is therefore far more likely that meningeal peaks A, B, G, and H were due to modifications within the vasculature rather thermore, we were unable to formylate our synthetic PA"", even under conditions which were far more extreme than than to contamination by parenchymal PA. those used to solubilize the amyloid cores. It is possible that DISCUSSION the formylation observed by others (34, 35) might have been In this study we demonstrate that PA purified from the induced by impurities in the commercial sources of formic brain parenchyma and leptomeningeal microvasculature con- acid, which we removed by redistillation. In agreement with earlier studies (1, 4, 7, 46), we observed tain structurally altered aspartyl residues, with positions 1 a variety of NH2 termini in parenchymal PA corresponding and 7 existing as a mixture of normal aspartyl and isoaspartyl isomers in both the D- and L-stereoconfiguration. This is the to residues Asp', Ala', Asp7,Sera, Gly', Tyr", and Phe". These first report of aspartyl isomerization yielding D- and L-isoas- were observed by amino acid composition analyses, sequencpartyl residues in PA (74, 75). Indeed, the major species of PA ing, and massspectroscopy. Whereas themajor species (Asp', purified from parenchymal amyloid corescontains anL-isoas- Ala', Sera, Gly', and Tyr") have been described previously(1, partyl residue at position 7. The more minor modification, 7, 46), this is the first report of minor species beginning at that of racemized residues, has been reportedbefore; Shapira Asp7 and PheIg. These were observed onlyin the mass spectral et al. (45) reported that theiramyloid preparations contained analyses andmay have been missed by less sensitive methods. previously by approximately 5% D-As~ and2% D-Ser. They did not, how- We did notobserve three NH2 termini reported ever, determine the sites of modification. The relative levels others, corresponding to Glu3 (4), Phe4 (1, 7 ) , and Va1I2 (8). we measured for D - A s ~(11%) and D-Ser (6.2%) in PA are These discrepancies might be due to methodological differsignificantly higher. This discrepancy may be due to the fact ences in PA purification or to heterogeneity among AD brains. In contrast to earlier reports (2-4), we have shown that the t h a t our core preparations were more highly purifiedor to the predominant parenchymal PA extends toAla42and that menwell known variabilityin numerous pathological features ingeal PA is COOH-terminally truncated at Val4'. One set of among AD brains. studies claimed that meningeal PA extends only to residue 39 The relative elution order of the Tp2 peptides by reverse phase HPLC was consistent with earlier studies. Ina similar (2, 3). We saw no evidence of tryptic peptides terminating at analysis of an unrelated peptide containing this combinationVal3' in any of our analyses. Since our purification method for meningeal PA did not differ appreciably from that used in (36), the peptide of aspartyl isoforms at asingleposition the earlier reports(2,3), we expect that thisdiscrepancy may containing L-iso-Asp eluted first,followed by those containing be due to the relative quality of the collagenase used during D-iso-Asp, L-ASP,and D-As~, respectively. The different eluPA purification. Amore recentstudy by Mori et al. (4) tion order for the Tpl peptides may have been due to the suggested that the most abundant@Ain AD brains is PA"". presence of the altered residues at the NH;?terminus. TheDOur data also fail to support this claim. These investigators Ser residues were fairly well distributed among the various utilized a C4 reverse phase column during their purification, /3A6-"jisoforms, hence their presence did not appear to affect likely resultingintheelimination of from theirprepathe chromatographic behavior of these peptides. The only rations (see "Results"). We, on the other hand, eliminated a other PA tryptic peptidewhich contains serine (PA"-'8) lacks soluble PA fraction which contained predominantly flA1-40, a n aspartyl residue. Since this peptide eluted from reverse the since our purification schemeexploited the insolubility of the phase columns as a unique peak, the presence of racemized parenchymal cores prior to size-exclusion the chromatography residues was not investigated. step. This soluble material never exceeded 15% of the total The degradation of aspartyl residues in PA likely occurs PA"", however, in multiple preparations of amyloidcores through the formation of a succinimide intermediate, followed from several lots of cerebral hemispheres. Hence, was by racemization and hydrolysis to produce the four isoforms clearly thepredominant PA speciesin the AD brains we observed in this study(see Fig. 5 and "Results"). This process utilized. has beenshown to occur at rates which are biologically Mori et al. (4) further suggested that @A"43accounted for relevant in peptide substrates under physiological conditions, 5-25% of their material. We observed a small quantity of with t l l P as short as41 days for L - A s ~isomerization and 19.5 /3A'9-43 in trypticdigests of only one parenchymal preparation, h forsuccinimide epimerization (36). The molar ratio of accounting for less than 5% of the total Tp4 in that sample isoaspartyl (peaks G and H) to normal aspartyl (peaksI and (not shown). This peptide was not observedin any other I-bis) residues at position 7 is about 3 to 1 (Table 11), which parenchymal or meningeal preparations. Since residue 43 is is essentially thesameratio seen instudies of synthetic Thr,shouldbe more hydrophilic than PA"", possibly peptides in which the aspartyl, isoaspartyl, andsuccinimidyl accounting for its recovery by Mori et al. (4) through the C4 forms are inequilibrium (36). The propensityfor succinimide column. Consistent with this,we have found that a synthetic cyclization may be in part dependent on the adjacent amino peptide which would correspondto PA residues 35-43 acid, beingmorefavored with a small side chain residue (VGGVVIAT) exhibits a shorter retention time on reverse COOH-terminal to theAsp in one study(37). This is consist- phase HPLC than /3A35-42(VGGVVIA) produced as a CNBr e n t with the isomerization observed in the tryptic peptides fragment of our synthetic PA"4' (not shown). Toexplain the T p l and Tp2, in which the Asp residues are followed by Ala discrepancies between these two studies, we considered the and Ser, respectively. The lack of isomerization at AspZ3 possibility that the DNase and collagenase employedin during within the Tp3 peptide may be explained by the adjacent our purification process might have generated PA"42 artifac-

3082

Structurally Altered

Asp Residues in Alzheimer &Amyloid

tually froma PA"43 ''precursor." This is unlikely for two all-D-substrate, failing to degrade the corresponding all+ reasons. First, our syntheticPA"42 peptide was unaffected by substrate (72). It has also been shown that carboxypeptidase prolonged incubation with these enzymes at levels greater y cannot efficiently cleave an isoaspartyl bond (27, 73). The than those used to treat our tissue samples (not shown). Any usual controls over a complement-mediated attack on PA- or contaminating proteolytic activity would need to have been PAPP-containing structures mightbe lost if abnormal asparspecificfor the single COOH-terminalthreonine. Second, tyl residues exist within a site which either binds a catabolic was the predominant species (85-95%) in all of our protease or serves as its substrate. Abnormal aspartyl residues preparations. Its derivationdue to proteolysis of would within thePA domain might also suppress the PAPP secretase beinconsistent with thepresence of as a minorfraction from clipping theprecursor,preservingtheintact PA se( 5 2 5 % ) in the report by Mori et al. (4).Hence, in the Mori quence. If PA depositsare responsiblefor activatingthe et al. study (4),the greater relative abundance of PA1-43 was complement cascade, this resulting stability could lead to a likelydue tothe loss of a prominentfraction.Inany relentless attack. This might explain why the neuronal induccase, it isdifficult to argue these points quantitatively due totion of the MIRL fails to save the affected neurites, as well the well known heterogeneity among AD brains. For example, as the resistanceof AD amyloid deposits to thesedegradative Mori et al. (4) found no trace of NHn-terminal Ala arising processes. Notably, although amyloid deposits contain prefrom PA residue 2, whereas we found low levels of this peptide dominantly PA"", such a scenario would not necessarily by sequence analysis and mass spectrometry. They also foundrequire a full-length /3A1-42peptide to nucleate an amyloid approximately 15-20% NH2-terminal pyroglutamate, arising deposit. from PA residue 3. We found no evidence for pyroglutamate How might a detrimental content of structurally altered at this position. In agreement with our study, Mori et al. (4) aspartyl residues accumulate in AD? It has been proposed also failed to detect PA peptides ending with Val3'. Hence if thattheL-isoaspartate(D-aspartate)0-methyltransferase they actually did characterize a predominantly vascular PA may be a repair enzyme which limits the accumulation of Lpreparation,our respective studies may be in fairly close iso-Asp a n d D - A s ~residues intracellularly (27, 42). A defiaccord. ciency in this type of corrective mechanism might be pathoThe reason for PA NH2- and COOH-terminal heterogeneitylogical, consistent with the elevated levels of D-As~ andLremains elusive. Considerable effort is being directed within iso-Asp foundin AD neurofibrillary tangles (74, 75). An the AD amyloid field toward identifying brain proteases which investigation of the overall activity of this enzyme in brain can cleave the PAPP with the specificity to liberate intact extracts, however, revealed no decrement in AD relative to It is thought that inhibition of such proteases might age-matched controls(76). In this context, the recently cloned mitigate the AD-associated amyloidosis. It remains unclear, cDNA of the human methyltransferase hasbeen used to map however, whether such specific mechanisms are necessary for the correspondinggene (77),and thischromosomal locus does senile plaque formation. For example, it is known that the not coincide with any which have been identified as relevant amyloidoticneuropil is a targetforcomplement-mediated to AD (78, 79). It is therefore unlikely that a deficiency in attack resulting ina lytic assault on the surrounding neuritesthis enzyme is responsible for AD-type pathology. (47,48). It has also beenreported that the membrane inhibitor Alternatively, since the greatest content of abnormal asof reactive lysis (MIRL) is induced by the affected neurons, partyl stereoconformers has been observed in long-lived proin an apparent compensatorysurvival attempt (49). Amyloid teins (80), the level of these altered residues in PA peptides deposits are nevertheless bathed in lysosomal proteases and might be affected by changes in the half-life of PAPP or its hydrolases thoughttoarisefromthe locally degenerating fragments. The elevated PAPP immunostaining within neuneurites (50-52), suggesting that the defense against comple- ritic terminals surroundingamyloid deposits (53-60) suggests that PAPP turnover is reduced in AD. This could lead to an ment attack fails in AD brain. Since PAPP accumulates in the neuritic terminals surrounding amyloid deposits (53-60), increase in abnormal stereoconformer contentintracellularly it is possible that longer segments of the PAPP are initially without any alteration in methyltransferase activity. Alternatively, PA peptides which contain abnormal aspartyl sterdeposited on an extracellular surface (61-66) and then proeoconformers may arise from PA-containing COOH-terminal teolyzed nonspecifically by the hydrolytic enzymes released PAPP fragments contained within the endosomal/lysosomal from the degenerating neurites. Missing from this scenario isa known source of intact PA system (14-16), since these would be sequestered from the domain. PAPP segments containing the intact PA domain cytoplasmic methyltransferase. An extracellular stabilization of amyloidogenic fragments have been identified within cultured cells (14, 67) and within AD brain (17). Some of these have been localized within the might also occur in AD brain, either through binding interactions of PA-containing PAPP fragmentswith extracellular lysosomal/endosomal system (14, 16), suggesting that they might be released along with other lysosomal constituents matrix proteins (61-66) or with incipient amyloid deposits following an initial neuritic insult. Intact PAPP might also be (69). In this case, most of the structural alterations might released from the degenerating neuritic terminals. This dy- occur after the initial deposition ofPA peptides or longer is, of course, important to emphasize that namic process would be expected to leave the PA peptide as PAPP fragments. It the longest lived PAPP segment due to its self-aggregating none of these proposed mechanisms are mutuallyexclusive. In summary,we have identified structural alterations in the properties and resistance toproteolytic attack (68), thus providing a template for further proliferation of the amyloid peptide backbone of amyloid core protein purified from AD brain tissue. These abnormal structures were shown to be plaque (69). present in the majority of PA peptides purified from amyloid Amyloidogenic mechanisms areknown to exist in the brains of nondemented individuals, although apparently under suf- cores of the brain parenchyma.It is possible that their presficient control to prevent the pathological consequences lead- ence is mechanistically involved in the induction and propaing to AD (70). The accumulation of abnormal aspartyl resi- gation of amyloid deposits in AD. 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