Identification of synaptic plasma membrane proteinsco-precipitated with fibrillar b-amyloid peptide

Yann Verdier,* Em}oke Huszar,* Botond Penke,*,� Zsuzsa Penke,� Gary Woffendin,§ MichaelaScigelova,§ Lıvia Fulop,* Maria Sz}ucs,¶ Katalin Medzihradszky** and Tamas Janaky*,�

*Department of Medical Chemistry, University of Szeged, Szeged, Hungary

�Protein Research Group of the Hungarian Academy of Sciences, Szeged, Hungary

�Department of Comparative Physiology, University of Szeged, Szeged, Hungary

§Thermo Electron, Hemel Hempstead, UK

¶Institute of Biochemistry, Biological Research Center, Szeged, Hungary

**Biological Research Center, Szeged, Hungary and Department of Pharmaceutical Chemistry, School of Pharmacy, University of

California, San Francisco, California, USA

Abstract

The b-amyloid peptide that is overproduced in Alzheimer’s

disease rapidly forms fibrils, which are able to interact with

various molecular partners. This study aimed to identify

abundant synaptosomal proteins binding to the fibrillar

b-amyloid (fAb) 1–42. Triton X-100-soluble proteins were

extracted from the rat synaptic plasma membrane fraction.

Interacting proteins were isolated by co-precipitation with

fAb, or with fibrillar crystallin as a negative control. Protein

identification was accomplished (1) by separating the trypti-

cally digested peptides of the protein pellet by one-dimen-

sional reversed-phase HPLC and analysing them using an

ion-trap mass spectrometer with electrospray ionization; and

(2) by subjecting the precipitated proteins to gel electropho-

retic fractionation, in-gel tryptic digestion and to matrix-

assisted laser desorption/ionization time-of-flight mass

measurements and post-source decay analysis. Six different

synaptosomal proteins co-precipitated with fAb were identi-

fied by both methods: vacuolar proton-pump ATP synthase,

glyceraldehyde-3-phosphate dehydrogenase, synapsins

I and II, b-tubulin and 2¢,3¢-cyclic nucleotide 3¢-phospho-

diesterase. Most of these proteins have already been asso-

ciated with Alzheimer’s disease, and the biological and

pathophysiological significance of their interaction with fAb is

discussed.

Keywords: Alzheimer’s disease, b-amyloid 1–42, glycolysis,

matrix-assisted laser desorption/ ionization time-of-flight mass

spectrometry, proteins interacting with b-amyloid, synaptic

plasma membrane.

J. Neurochem. (2005) 94, 617–628.

b-Amyloid (Ab), which is overexpressed in Alzheimer’sdisease (AD), accumulates extracellularly in neuritic plaquesand aggregates intracellularly (Buckig et al. 2002). Ab is apeptide of 39–43 amino acids, which is generated by multipleproteolytic processing of a large transmembrane precursor,the amyloid precursor protein (APP). By virtue of itsstructure and its physicochemical properties, Ab is able tobind a wide variety of biomolecules, including lipids,proteins and proteoglycans (for a review, see Verdier et al.2004). This binding may interfere with various biologicalprocesses, and it is well established that Ab inducesneurotoxicity both in vitro and in vivo (for review, seeHarkany et al. 2000). In AD, loss of synapses exceedsneuronal loss, and the synaptic pathology is characterized byimpairment of synaptogenesis and neuronal transmission,

Received November 12, 2004; revised manuscript received February 4,2005; accepted February 4, 2005.Address correspondence and reprint requests to Yann Verdier,

Department of Medical Chemistry, University of Szeged, Dom ter 8,H-6725 Szeged, Hungary. E-mail: yverdier@hotmail.comAbbreviations used: Ab, b-amyloid; AD, Alzheimer’s disease; APP,

amyloid precusor protein; CID, collision-induced dissociation; CNPase1,2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase 1; fAb, fibrillar b-amyloid;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LC–MS/MS,liquid chromatography–mass spectrometry/mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight massspectrometry; PSD, post-source decay; SDS–PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis; SPM, synaptic plasmamembrane; TEM, transmission electron microscopy; V-ATPase C,vacuolar proton-pump ATP synthase subunit C.

Journal of Neurochemistry, 2005, 94, 617–628 doi:10.1111/j.1471-4159.2005.03158.x

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 617–628 617

synaptosomal loss and derangement of synaptosomal pro-teins, suggesting the activation of synaptic apoptotic mech-anisms (Yoo et al. 2001; Mungarro-Menchaca et al. 2002).

It has recently been suggested that an intrinsic Ab pathwaymay be as important as an extrinsic one in AD (Dickson2004). Intracellular accumulation of Ab is supposed tooriginate from APP processing from subcellular organelles,including the endoplasmic reticulum/intermediate compart-ment (Hartmann et al. 1997), the trans-Golgi network (Xuet al. 1997) and the endosomal/lysosomal system (Koo andSquazzo 1994), as well as from cellular uptake of extracel-lular Ab (Ida et al. 1996). Intraneuronal Ab 1–42 has beenlinked to dysfunction of mitochondria (Busciglio et al.2002), lysosomes (Glabe 2001) and synapses (Takahashiet al. 2002), and has also been related to cytosolic p53-associated cell death (Ohyagi et al. 2005).

Conversion of Ab from a soluble to a fibrillar formmarkedly increases its binding to membrane lipids andproteins. Ab oligomers and protofibrils have been implicatedin neurotoxicity through their direct action on neuronal cells.It has been proved that fibrillar (f)Ab also binds neuronalmembranes and has neurotoxic effects (Blanchard et al.2004) including neuronal dystrophy (Grace and Busciglio2002; for reviews see Selkoe 2002; Verdier et al. 2004).

The different forms of Ab bind various membrane proteins.Both themonomeric and thefibrillar formofAb are able to bindthe a7 nicotinic acetylcholine receptor (Wang et al. 2000),integrins (Sabo et al. 1995), the receptor for advancedglycosylation end-products (Yan et al. 1996a) and the formylpeptide receptor-like 1 (Yazawa et al. 2001). The fibrillar formof Ab has been reported to bindAPP (Lorenzo et al. 2000), theN-methyl-D-aspartate receptor (Bi et al. 2002), the P75neurotrophin receptor (Yaar et al. 1997), the collagen-likeAlzheimer amyloid plaque component precursor/collagenXXV (Hashimoto et al. 2002), the scavenger receptors A,BI (El Khoury et al. 1996) and CD36 (Coraci et al. 2002),and a complex involving CD36, a6b1-integrin and CD47(Bambergeret al. 2003). The binding ofAb to thesemembraneproteins induces various beneficial effects, such as Ab clear-ance, and also negative effects, including internalization of Abor of the binding protein, and generation of toxic free radicals(for reviews see Verdier and Penke 2004; Verdier et al. 2004).

The binding of Ab to the plasma membrane is a potentialpoint of intervention for slowing down or preventing thedevelopment of AD. Thus, there is a growing interest in theidentification of Ab-binding membrane proteins. Moreover,the identification of synaptosomal molecular partners of Abshould allow a better understanding of AD. Interaction studiesbetween Ab and membrane proteins usually focus on a proteinalready implicated in AD. For example, the a7 nicotinicacetylcholine receptor has been studied because of the vulner-ability of cholinergic neurones in AD (Wang et al. 2000), andAPP–Ab interaction has been analysed with anti-APP anti-bodies owing to the role of APP in AD (Lorenzo et al. 2000).

Excellent results have been obtained using these approaches;however, the role of other proteins is still underestimated. Theaim of the present study was to identify the membrane ormembrane-associated synaptosomal proteins interacting withfAb using co-precipitation as a systematic approach.

To achieve this goal, Triton X-100-soluble proteins wereextracted from a rat synaptic plasma membrane (SPM)fraction. The interacting proteins were co-precipitated withfAb, or with fibrillar crystallin as a negative control. Proteinidentification was accomplished (1) by analysing the trypticdigest of the protein pellet by reversed-phase HPLC coupledwith electrospray ionization and collision-induced dissoci-ation in a data-dependent manner; and (2) by subjecting theprecipitated proteins to gel electrophoretic fractionation, in-gel tryptic digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) mass measurements and post-source decay (PSD)analysis. Although liquid chromatography–mass spectrome-try/mass spectrometry (LC–MS/MS) analyses identifiedseveral proteins that selectively co-precipitated with fAb,only six such components could be unambiguously identifiedafter sodium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS–PAGE) fractionation.

Materials and methods

Materials

Ab 1–42 (DEAFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLM

VGGVVIA) was synthesized in our laboratory on a solid-phase

support, as described in Palotas et al. (2002). After purification, itwas lyophilized from aqueous solution. Repeating this operation

twice from water resulted in Ab 1–42 with mainly protofibrillar and

fibrillar structure. b-Crystallin and P-8340 protease inhibitor

cocktail were purchased from Sigma (St Louis, MO, USA).

Peptide preparation and transmission electron microscopy

Ab 1–42 was dissolved in water at 66 lM and incubated for 48 h at

37�C. b-Crystallin was dissolved in 10 mM Tris HCl, pH 6.8, at

200 lM (Meehan et al. 2004) and incubated for 48 h at 37�C. Thestructure of the peptide aggregates was checked by transmission

electron microscopy. Some 10 lL of the aggregated peptide sample

was adsorbed on to 400-mesh carbon-coated copper grids (Electron

Microscopy Sciences, Washington, PA, USA), fixed with 0.5%

(w/v) glutaraldehyde solution, washed three times with water and

finally stained with 2% (w/v) uranyl acetate. Specimens were

studied using a Philips CM 10 transmission electron microscope

(FEI company, Hillsboro, OR, USA) at 100 kV. Images were taken

with a MegaView II Soft Imaging System (GmbH, Munster,

Germany) at a magnification of · 46 000.

Purification of the subcellular synaptosomal fractions

Subcellular fractionation of rat brains (four male Wistar rats, 200–

250 g) was performed as described by Fabian et al. (2002). Briefly,fresh forebrains were gently homogenized. The homogenates were

centrifuged twice at 1000 g, and the combined supernatants were

618 Y. Verdier et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 617–628

centrifuged at 20 000 g for 20 min. Pellets were suspended in 10%

buffered sucrose and subjected to consecutive centrifugations at

20 000 g for 25 min and twice at 14 000 g for 20 min. The resulting

pellets were lysed by Dounce homogenization and incubation for

30 min on ice in lysis buffer (0.05 mM CaCl2, 0.5 mM dithiothreitol,

50 mM Tris-HCl, pH 8.1), followed by fractionation on a 10/28.5/

34% sucrose density step gradient that was centrifuged at 128 000 gfor 2 h. Highly enriched SPMs were obtained from the 28.5/34%

interface. The SPM fraction was diluted threefold with 50 mM Tris-

HCl, pH 7.4, and pelleted at 128 000 g for 1 h.

Co-precipitation binding assay

This assay was performed with some modifications of the method

described by Lorenzo et al. (2000). The SPM pellet was resuspended

in 4 mL 1% Triton X-100 in phosphate buffer, pH 6.8, containing

protease inhibitor cocktail, and centrifuged at 16 000 g for 30 min.

The supernatants containing the detergent-soluble proteins were

incubated overnight with vehicle or with fibrillar proteins (final

concentrations: 20 lM crystallin, 20 lM Ab). The samples were then

centrifuged at 16 000 g for 10 min, washed twice with 20 mM

HEPES, pH 7.0, and resuspended in 1% sodium azide in water.

LC–MS/MS identification of the proteins co-precipitated with

fAb

In-solution tryptic digestionDisulphide bridges in 25-lg samples of precipitated plasma

membrane were reduced with 7.5 lL 10 mM dithiothreitol and

alkylated with an excess amount of iodoacetamide (20 lL of

55 mM) in 75 lL buffer containing 6 M guanidine hydrochloride,

10 mM EDTA and 0.1 M Tris HCl, pH 8.0. The reaction mixture

was dialysed against 50 mL 1 M guanidine hydrochloride in 50 mM

ammonium bicarbonate solution, pH 8.0, then the proteins were

digested for 5 h at 37�C with side-chain protected porcine trypsin

(Promega, Madison, WI, USA) using an enzyme to protein ratio of

1 : 50 (w/w). The resulting total protein digest was desalted on a

reverse-phase column (Nucleosil 5, C18, 1 · 10 mm) and peptides

were eluted with 80% acetonitrile containing 0.1% formic acid.

LC–MS/MSThe above digests were analysed using a nanoflow HPLC system

Surveyor connected to an LTQTM linear ion trap mass spectrometer

equipped with nanoelectrospray ionization source (Thermo Electron,

San Jose, CA, USA). Some 2 lL (1 lg) of sample was loaded on to

a reverse-phase poly(styrene-divinylbenzene) peptide trap (Michrom

BioResources, Auburn, CA, USA) at a flow rate of 10 lL/min. The

peptides were eluted from the trap directly on to a reverse-phase

nanocolumn [75 lm · 10 cm PicoFrit column (New Objective,

Woburn, MA, USA) packed with 3 lm Biobasic C18, 0.3 lm pore

size packing material, Thermo Electron]. A linear gradient of 0–60%

of solvent B in solvent A B was developed over 60 min at a flow

rate of approximately 200 nL/min. Solvent A was 0.1% formic acid

in water. Solvent B was 0.1% formic acid in 100% acetonitrile. The

spray voltage was 2.0 kV and the capillary temperature was 150�C.The collision-induced dissociation spectra were acquired at collision

energies of 35%. Data were acquired in a data-dependent fashion:

each mass acquisition was followed by three zoom-scans and three

collision-induced dissociation (CID) experiments performed on the

computer-selected three most abundant ions of the mass measure-

ment experiments. Singly charged ions were not analysed by CID.

The dynamic exclusion feature was also activated; ions once

subjected to CID analysis were excluded from the precursor ion

selection process for 60 s.

Data analysisProtein identification was performed using the MASCOT algorithm

(http://www.matrixscience.com) and the Swiss-Prot rat database

(Swiss Institute of Bioinformatics, Geneva, Switzerland) with

carbamidomethyl Cys as fixed modification; protein N-acetylation,oxidation of Methionine and pyroglutamate formation from Gln

were allowed as variable modifications; mass accuracy was

considered within 3 Da for MS and 0.8 Da for MS/MS. Only

proteins with at least two matching sequences were considered. The

same database was also screened using the Turbo-SEQUEST

algorithm in the BioWorks 3.1 software package (Thermo Electron).

Gel electrophoresis, in-gel digestion and identification by mass

spectrometry of the proteins co-precipitated with fAbSamples containing 10–30 lg protein were separated by SDS–PAGEand visualized by colloidal Coomassie Brilliant Blue G-250 using

standard protocols (Laemmli 1970; Wu and Welsh 1996). Protein

bands that co-precipitated with fAb were cut. The Coomassie stain

and SDS were removed by repeated washing with 25 mM ammonium

bicarbonate, pH 8.1, in acetonitrile : water 1 : 1mixture. Disulphides

were then reduced with 10 mM dithiothreitol and the free -SH groups

were alkylated with 55 mM iodoacetamide. The reagent excess was

removed by repeated washing with 25 mM ammonium bicarbonate in

acetonitrile : water 1 : 1 mixture. Proteins were in-gel digested with

side-chain protected porcine trypsin (Promega) for 4–6 h at 37�C(http://donatello.ucsf.edu/ingel.htm). Tryptic peptides were then

extracted from the gel with a 5% formic acid solution in acetonit-

rile : water 1 : 1 mixture. The volume of the extracted digests was

reduced to 10 lL using a Speed Vac and the mixtures were desalted

on C18 ZipTip (Millipore, Bedford, MA, USA), according to the

manufacturer’s instructions. A 0.5-lL aliquot of each sample was

mixed with 0.5 lL freshly prepared 2,5-dihydroxybenzoic acid

matrix solution [saturated solution in 0.1% (v/v) trifluoroacetic acid

in water] directly on the target, and then air dried. Mass analyses were

performed on a Bruker Reflex III MALDI-TOF mass spectrometer

(Bruker, Leipzig, Germany) in positive reflecton mode. Spectra were

calibrated externally using a set of peptide standards. The full

National Center for Biotechnology Information) non-redundant

protein database was searched with the monoisotopic MH+ values

determined using on-line software search algorithms MASCOT

(http://www.matrixscience.com) and Protein Prospector (http://

www.prospector.ucsf.edu). To confirm the results of the mass

fingerprint-based database searching, PSD spectra of selected

peptides were recorded in order to gain sequence information.

Results

Structure of the aggregates used for the co-precipitation

assay

The structure of Ab 1–42 and b-crystallin aggregates wasstudied by transmission electron microscopy. In the time

Proteins co-precipitated with b-amyloid 619

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 617–628

course of the biological experiments both samples containedfibrillar structures, but with a different morphology (Fig. 1).Fibrillar b-crystallin was found to contain spherical aggre-gates with an average diameter of 20 nm, as well as chain-like structures a maximum of 800 nm long with theappearance of row of beads of diameter 15–25 nm. Thisphenomenon is typical of the self-assembled specimens ofthe crystallin classes as demonstrated by Meehan et al.(2004). On the contrary, Ab 1–42 formed protofibrils 3–4 nmwide and 5–70 nm long, and smooth, non-branched maturefibres of diameter 7–10 nm and maximum length severalmicrometres, in accordance with the generally acceptedaggregation process of the peptide.

LC–MS/MS identification of the proteins co-precipitated

with fAbTriton X-100-soluble proteins extracted from the SPM of ratbrains were precipitated with fAb, fibrillar crystallin or withwater and, after washing, the co-precipitated proteins weresubjected to tryptic digestion. The resulting peptides wereanalysed by LC–MS/MS (Fig. 2), and the correspondingproteins were identified by performing a rat-specific databasesearch with all the CID data acquired against the SwissProtprotein database.

Eighty-eight proteins were identified as being co-precipita-tedwith fAb, including 53 proteins identified from twoormorepeptide sequences and a MASCOT score > 44. To reduce theprobability of false-positive results, only these 53proteinsweretaken into consideration. However, 13 of these proteins werealso detected in the control experiment, i.e co-precipitatedwiththe crystallin. These were: c-actin (Accession numberP63259), b-actin (P60711), guanine nucleotide-binding pro-tein Go a1 (P59215), Thy-1membrane glycoprotein precursor(P01830), spectrin a chain (P16086), contactin precursor(Q63198), neuronal growth factor precursor (Q9Z0J8),synaptosomal-associated protein 25 (P60881), carbonicanhydrase III (P14141), Abl-interactor 1 (Q9QZM5), limbicsystem-associated membrane protein precursor (Q62813),b-crystallin B2 (P62697) and Noelin precursor (Q62609).

Table 1 shows the 40 proteins that co-precipitated withfAb identified in this experiment (from two or more uniquepeptides and a significant MASCOT score > 44). All theseproteins were also identified from the same database usingthe Turbo-SEQUEST algorithm in the BioWorks 3.1 soft-ware package (Thermo Electron).

Fig. 1 Fibrillar structure of b-crystallin and Ab 1–42 used for the

precipitation assay, assessed by transmission electron microscopy.

(a) b-Crystallin dissolved at 200 lm in 10 mM Tris, pH 6.8, was incu-

bated for 48 h at 37�C. The peptide formed spherical aggregates as

well as chain-like structures which appeared as rows of beads of

diameters 15–25 nm with a maximum length of 800 nm. (b) Ab 1–42

dissolved at 66 lM in water was incubated for 48 h at 37�C. Protofibrils

3–4 nm wide and 5–70 nm long, and smooth fibres of diameter

7–10 nm and a maximum length of several micrometres were present

in the sample. Magnification · 46 000.

Fig. 2 MS/MS spectrum of a peptide that

eluted with a retention time of 28.57 min.

Precursor mass is 2051.97. Rat synapsin86QTTAAAATFSEQVGGGSGGAGR108

was identified from these data.

620 Y. Verdier et al.

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 617–628

SDS–PAGE fractionation of the proteins co-precipitated

with fAb, and protein identification by MALDI-TOF MS

and PSD

As we could not draw reliable conclusions about the quantityof the precipitated proteins by LC–MS/MS, and in order to

confirm the identity of the proteins by another method,proteins precipitated with fAb or with fibrillar crystallin werefractionated by SDS–PAGE on an 8% acrylamide gel. AfterCoomassie blue staining, only 18 protein bands werevisualized in the fAb sample, including 11 bands also

Table 1 Proteins identified in data-

dependent LC–MS/MS experiments (at

least two matching peptides)Name

Accession

no.

Peptides

matched Score

Synapsin-1 P09951 73 2522

Vacuolar ATP synthase subunit B, brain isoforms

(EC 3.6.3.14)

P62815 46 1446

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) P04797 34 1351

Bassoon protein O88778 41 1030

Synapsin-2 Q63537 22 873

Tubulin b-chain P04691 25 825

Myelin basic protein S P02688 32 769

Vacuolar proton translocating ATPase 116-kDa subunit

a isoform 1

P25286 31 750

p130Cas-associated protein Q9QXY2 12 442

Tubulin a-1 chain P02551 16 433

2¢,3¢-Cyclic nucleotide 3¢-phosphodiesterase (EC 3.1.4.37) P13233 12 396

b-Amyloid A4 protein precursor P08592 5 279

c-Enolase (EC 4.2.1.11) P07323 3 237

Na+/K+potassium-transporting ATPase a-3 chain (EC 3.6.3.9) P06687 7 226

Elongation factor 1-a 1 P62630 7 215

Brain acid-soluble protein 1 Q05175 3 201

Dihydropyridine-sensitive l-type, calcium channel

a-2/d subunit precursor

P54290 6 164

Synapsin-3 O70441 5 157

Flotillin-1 Q9Z1E1 7 156

Septin 7 Q9WVC0 7 139

Neurotrimin precursor Q62718 3 128

Vesicle-associated membrane protein 2 P63045 2 127

Cofilin, non-muscle isoform P45592 3 122

Succinyl-CoA ligase [GDP-forming] a-chain, mitochondrial

precursor (EC 6.2.1.4)

P13086 5 118

ATP synthase a chain, mitochondrial precursor (EC 3.6.3.14) P15999 5 117

Piccolo protein Q9JKS6 9 117

Microtubule-associated protein 2 P15146 3 117

Regulating synaptic membrane exocytosis protein 1 Q9JIR4 2 89

Ras GTPase-activating protein SynGAP Q9QUH6 3 88

ATP synthase e chain, mitochondrial (EC 3.6.3.14) P29419 2 88

Opioid-binding protein/cell adhesion molecule precursor P32736 5 87

Cytochrome c oxidase subunit IV isoform 1, mitochondrial

precursor (EC 1.9.3.1)

P10888 3 69

Ras-related C3 botulinum toxin substrate 1 Q6RUV5 4 68

Contactin-associated protein 1 precursor P97846 3 67

Rabphilin-3A P47709 8 66

Gap junction a-1 protein P08050 3 61

Contactin 2 precursor P22063 3 59

Presynaptic density protein 95 P31016 4 52

ELKS-Rab6-interacting protein-Cytomatrix at the active

zone associated structural protein 2

Q8K3M6 3 48

Calcium/calmodulin-dependent protein kinase type II a chain

(EC 2.7.1.123)

P11275 3 44

Entries in bold were also identified by SDS–PAGE fractionation followed by in-gel digestion and

MALDI-TOF MS analysis. All these proteins precipitated with fAb but not with fibrillar crystallin.

Proteins co-precipitated with b-amyloid 621

� 2005 International Society for Neurochemistry, J. Neurochem. (2005) 94, 617–628

detected in the crystallin precipitation (Fig. 3a). Figure 3(b)shows the reproducibility of the procedure, from subcellularfractionation to electrophoretic analysis; the same bands wereobtained in separate experiments.

The seven proteins selectively co-precipitated with Abwere subjected to in-gel tryptic digestion, and the digestswere analysed by MALDI-TOF MS (Fig. 4a). PSD analysesof selected components were also performed (Fig. 4b).Table 2 shows the proteins identified in these experiments.

Discussion

Ab that is overproduced in AD rapidly forms fibrils, whichare able to interact with various molecular partners. Althoughseveral studies using different methodologies have identifiedproteins that bind fAb, some of the molecular partners of fAbremain unidentified. This study aimed to identify synapto-somal (membrane, membrane-associated and other) proteinsthat interact with fAb.

In order to isolate and identify rat forebrain SPM proteinsthat interact with fAb, we developed a co-precipitation assay.Subcellular fractionation reduces the complexity of thesample and was used to increase the proportion of membraneproteins in the mixture co-precipitated with fAb. It has beenreported that the sucrose density gradient method producesthe highest membrane protein concentration compared withother protein isolation techniques (Roth et al. 1981). Thismethod has been used for the identification of SPM proteinsalso found in the present report, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), synapsins I and II, Na+/K+ transporting ATPase a, the opioid-binding protein,bassoon, a protein similar to SynGAP, septin 7, the gap

181716

15

14

131211

1098765

43

21

18

1 2 3 4

171615

14

131211

109876543

21

97 kDa

66 kDa

45 kDa

31 kDa

21 kDa

97 kDa

66 kDa

45 kDa

31 kDa

21 kDa

+ w

ater in

tra-te

stre

peat

abili

tyin

tra-te

stre

peat

abili

ty

+ cr

ysta

llin

(20

µM)

+ cr

ysta

llin

(2 µ

M)

+ A

β (2

0 µM

)

(a) (b)

Fig. 3 (a) Electrophoretic analysis of the proteins precipitated with

water, 20 lM fibrillar crystallin, 2 lM fibrillar crystallin and 20 lM

fibrillar Ab 1–42, on an 8% acrylamide Coomassie blue-stained gel.

Bands number 3, 5, 7, 8, 9, 12 and 13 were specifically precipitated

with fibrillar Ab 1–42. (b) Reproducibility of the precipitation assay

with fibrillar Ab 1–42 assessed on a 12% acrylamide Coomassie

blue-stained gel. Proteins extracted the same day were incubated

with Ab 1–42 in different tubes (intratest reproducibility, lanes 1 and

2). To assess the intertest reproducibility, synaptosomal extraction,

protein purification and precipitation with Ab 1–42 were done on dif-

ferent days (lanes 3 and 4). The same bands were detected on each

occasion.

1300 1800 2300 2800 m/z

0

2

4

6

8

10

12

14

a.Ix10.

*

*

*

*

*

*

* *

*

**

****

**

*

PSD

100 150 200 250 300 350 400 m/z

2

4

6

8

10

12

14

16

18

a.i.x10-2

R V

VG

V GV

NG

KG

V

PSD spectrumParent ion: 1032.61

84.2

0

129.

29

158.

28

175.

34

210.

32 215.

36 228.

3823

2.34

256.

8427

1.21

285.

39

313.

3532

8.24

356.

7136

2.88

384.

73

412.

56

436.

43

2933

.49*

2765

.5826

11.4

525

95.4

5

*

2369

.31

2347

.31

2333

.27

2293

.16

2266

.38

2277

.18

2213

.15

2127

.25

* *

1795

.78

1779

.78

1762

.82

1697

.8616

45.8

816

61.8

516

27.9

215

70.7

815

66.7

5*

1499

.74

1485

.66

1447

.74

1391

.67*

1369

.88

1356

.60

*13

31.6

0

1227

.56

1215

.63

1052

.51

1032

.61

934.

8889

1.5086

9.53

842.

56

70.1

872

.17

112.

2612

0.33

RG

V*

y1*

y1

y2*

b2*

VN

y4

b4

y3y3*b3

b3*

GV

N,V

GN

y2

b2

K

(a) (b)

Fig. 4 (a) MALDI-TOF mass spectrum of

the tryptic digest of band 3. The peaks

labelled with an asterisk correspond to

predicted tryptic peptides of the GAPDH

(P04797). Monoisotopic masses are dis-

played. (b) PSD spectrum acquired from the

precursor ion at m/z 1032.6. The confirmed

peptide sequence is 2VKVGVNGFGR11.

Fragment ions are labelled according to the

nomenclature of Biemann (1990).

622 Y. Verdier et al.

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junction a-1 protein, the synapse-associated protein SAP90,and calcium/calmodulin-dependent protein kinase II (Stevenset al. 2003). The use of synaptosomes should allow theidentification of biologically significant interactions, as Abhas been described as a synaptotoxic protein (for review, seeSelkoe 2002). The co-precipitation assay used in the presentwork enables the isolation of proteins directly bound to Aband of those that interact with other Ab-binding proteins.

Forty proteins that selectively interacted with fAb wereidentified by LC–MS/MS. A peptide with a sequencecommon to Ab 1–42 and APP was identified in the proteindigest; this was probably partly derived from the added Ab1–42 (similarly, crystallin was identified in the negativecontrol). Owing to the position and sequence of the identifiedpeptides, the possibility that the full APP was present in oursample cannot be dismissed (in accordance with the results ofa similar precipitation assay developed by Lorenzo et al.2000). Proteins co-aggregated with fAb could be sorted intosix groups according to their potential biological significancein AD as follows.

(1) Proteins with properties that can be linked with AD.The mitochondrial ATPase a has been associated withneurofibrillary degeneration in AD (Sergeant et al. 2003),translation elongation factor binds acetylcholine receptor(McClatchy et al. 2002), calcium/calmodulin-dependent pro-tein kinase II phosphorylates APP (645–661) (Gandy et al.1988), rabphilin-3A is involved in neurotransmitter exo-cytosis and its function is impaired after interaction witha-synuclein (Dalfo et al. 2004), and the amount of myelinbasic protein is decreased in AD (Roher et al. 2002).

(2) Proteins involved in cytoskeletal organization, whichdegenerates in AD (for review, see Harkany et al. 2000;Selkoe 2002). These proteins are: bassoon (Dresbach et al.2003), p130Cas-associated protein (Chin et al. 2000), cofilin(Minamide et al. 2000), piccolo protein (Fenster et al. 2000),

microtubule-associated protein 2 (Kindler et al. 1990) andseptin 7 (Kinoshita and Noda 2001).

(3) Neuronal cell adhesion molecules: contactin 1(Hosoya et al. 1995), contactin-associated protein 1 precur-sor (Peles et al. 1997), flotillin-1 (Lang et al. 1998),neurotrimin (Struyk et al. 1995), opioid-binding cell adhe-sion molecule (Miyata et al. 2003), gap junction a-1 protein(Dupont et al. 1991), contactin 2 precursor (Furley et al.1990) and ELKS-Rab6-interacting protein-Cytomatrix at theactive zone associated structural protein (ERC) protein 2(Ohtsuka et al. 2002). It has been suggested that celladhesion molecules such as integrins bind to Ab and playan important role in AD (for review, see Verdier et al. 2004).

(4) Other neuronal membrane proteins: brain acid-solubleprotein 1 (Epand et al. 2003), dihydropyridine-sensitiveL-type calcium channel (Kim et al. 1992), vesicle-associatedmembrane protein 2 (Elferink et al. 1989), regulatingsynaptic membrane exocytosis protein 1 (Wang et al. 1997)and Ras-related C3 botulinum toxin substrate 1.

(5) Neurone-specific proteins: c-enolase, Na+/K+ trans-porting ATPase a-3 subunit, synaptic Ras-GTPase-activatingprotein 1 and presynaptic density protein 95.

(6) Mitochondrial proteins: succinyl-CoA ligase, ATPsynthase e chain and cytochrome c oxidase subunit IV.Mitochondrial proteins are probably under-represented in ourresults because we selected the SPM fraction. It has beendemonstrated that mitochondrial dysfunction induced byAb isassociated with synapse loss and apoptosis in AD (Mungarro-Menchaca et al. 2002; for review, see Hashimoto et al. 2003).

These results suggest an interaction between fibrillar Aband intracellular proteins. Fibrils of Ab have been shown tointeract with a wide range of soluble proteins (Konno 2001).It has been proposed (Konno 2001) that, following theinteraction between fAb 25–35 and soluble proteins, theproteins might form amorphous aggregates and precipitate

Table 2 fAb-binding proteins identified by SDS–PAGE fractionation followed by in-gel digestion and MALDI-TOF MS analysis

Band Name

Accession

no.

MWExp

(kDa)

MWTheor

(kDa)

Fit

(%)

Coverage

(%)

Sequence justification

by PSD

3 Glyceraldehyde-3-phosphate dehydrogenase

(EC 1.2.1.12)

P04797 35.7 36.1 63 61 2VKVGVNGFGR11

5 Vacuolar ATP synthase, subunit C (EC 3.6.3.12) Q9Z1G3 41.7 44.0 44 33 2 TEFWLISAPGEK13

7 2¢,3¢-Cyclic nucleotide 3¢-phosphodiesterase 1

(EC 3.1.4.37)

P13233 46.7 47.6 42 34 391AIFTGYYGK399

8 2¢,3¢-Cyclic nucleotide 3¢-phosphodiesterase 1

(EC 3.1.4.37)

P13233 48.7 47.6 28 10 391AIFTGYYGK399

9 Tubulin b-5 chain P69897 52.3 50.0 38 40 31 LAVNMVPFPR 40

12 Synapsin-2 Q63537 72.3 64.0 32 40 178 SFRPDFVLIR 187

13 Synapsin-1 P09951 74.4 74.1 45 26 177SLKPDFVLIR186

Numbers refer to gel bands shown in Fig. 2(a). Theoretical MW (MWTheor) is calculated for the full sequence listed in the database that may not

reflect the real size of the mature/active protein. All these proteins precipitated with fAb but not with fibrillar crystallin. Experimental MW (MWExp

was determined by gel electrophoresis.

Proteins co-precipitated with b-amyloid 623

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together with amyloid fibrils. It is also possible thatunfolding of proteins induced by amyloid fibrils initiatesthe aggregation and precipitation of the proteins, because aprotein in an unfolded conformation has a much strongertendency to aggregate than one in a folded conformation(Fink 1998). Protein unfolding would also cause a strongadhesion of the protein on the amyloid fibril (Konno 2001).Both mechanisms, originally proposed for fAb 25–35, mightbe also valid for fAb 1–42 used in the present work. Thephysiological implication of this interaction is that amyloidfibrils might damage a wide range of biological systems byinteraction with extracellular as well as intracellular molecu-lar partners. However, the possibility should also be consid-ered that the co-precipitation of proteins with amyloid fibrilshas a beneficial physiological role, because adhesion of theproteins on the surface of amyloid fibrils might alsosubstantially alter the surface properties of these fibrils,resulting in loss of their cytotoxicity (Konno 2001).

The binding of fibrillar Ab to many intraneuronal andsynaptosomal proteins raises the possibility that interactionbetween Ab and these proteins might occur in vivo. Thepresent work focused on the interaction of fibrillar Ab andneural membrane proteins, as fibrillar Ab is present mainly inthe extracellular space of neural tissue. However, Ab isinternalized and fibrillized by macrophages in vivo andmicroglia in vitro (Yazawa et al. 2001; Nagele et al. 2004);moreover, there are indications that in special cases fibrillarAb can occur in neurones as well (Torp et al. 2000. Electronmicroscopic autoradiography studies in our laboratory, usingtritium-labelled oligomeric and fibrillar Ab 1–40 (in vivostudies on cat brain), showed that the labelled Ab enters theneurones in 30 min, possibly by an endocytotic pathway(Penke, B. unpublished results). On the other hand, Ab formsmainly cytosolic aggregates in neurones, distinct fromaggregasomes and fibrils (Buckig et al. 2002). Although thebinding properties of Ab aggregates and fAbmay be different,the possibility remains and should be explored that proteinsbound to fibrillar Ab also bind to the non-fibrillar form.

As we could not draw reliable conclusions by LC–MS/MSabout the quantity of the precipitated proteins, and in order toconfirm the protein identities by another method, we alsoanalysed these samples by SDS–PAGE followed by in-geldigestion and MALDI-TOF MS analysis. The seven mostabundant protein bands specifically co-precipitated with Ab(Fig. 3; corresponding to six proteins) were successfullyidentified (Table 2) and are discussed here in more detail.

The co-precipitation of the SPM GAPDH with fAb is themost significant result of the present report (Fig. 3a, band 3).Moreover, GAPDH extracted from cytosolic and microsomalfractions was also co-precipitated with 20 lM fAb andidentified by MALDI TOF (data not shown). GAPDH has,apart from its glycolytic role, various membrane, cytoplasmicand nuclear functions. Glycolysis is of special importance inneurones because of their high energy needs; reduction in

glucose turnover precedes cognitive dysfunction and istherefore believed to be an important early event in ADdevelopment (Arias et al. 2002). GAPDH has been reportedto interact with the C-terminal region of APP but not with Ab(Schulze et al. 1993), whereas another study reported aninteraction between monomeric Ab and GAPDH (Oyamaet al. 2000). Differences between these two studies areattributed to different Ab immobilization methods. In cellsaffected by AD, it has been demonstrated that APP bindingto GAPDH reduces subcellular GAPDH activity (Mazzolaand Sirover 2001). Binding between GAPDH and neuronalproteins may affect not only energy production but also othernon-glycolytic functions of GAPDH, including membranefusion and endocytosis, cytoplasmic mRNA regulation andmicrotubule bundling, and nuclear tRNA export, DNAreplication and DNA repair (for review, see Sirover 1999).Interestingly, GAPDH has been associated with variousneurodegenerative disorders, as it can bind proteins implica-ted in neuronal diseases, e.g. APP and huntingtin; isco-localized with a-synuclein in Lewy bodies; may functionas a pro-apoptotic protein in neuronal cells; and has aspecifically reduced subcellular activity in AD and Hunt-ington’s disease (for review, see Mazzola and Sirover 2002;Mazzola and Sirover 2003). It has been proposed that theformation of complexes of GAPDH and proteins implicatedin neuronal diseases may provide a molecular mechanismunderlying the common phenotypes (hypometabolism, apop-tosis) of neurodegenerative disorders, even if this may simplybe an epiphenomenon or a secondary effect (Mazzola andSirover 2002). Co-precipitation of GAPDH and fAb asshown the present report could therefore be of crucialimportance in AD.

Our results also showed that b-tubulin is co-precipitatedwith fAb. Tubulin is a microtubule subunit protein found inlarge quantities in the mammalian brain. It consists ofmultiple functional domains with distinct properties that aremediated by the binding of numerous agents, including ATP,GTP, microtubule-associated proteins and heat-shock pro-teins. Tubulin has been identified as a membrane componentof nerve synaptosomes and various plasma membranes.Tubulin serves as an attachment site for binding vesicles orplasma membranes to cytoplasmic microtubules, and inter-acts with various membrane proteins such as synapticmembrane G proteins (Yan et al. 1996b) and the a1metabotropic glutamate receptor (Ciruela et al. 1999).Modifications of b-tubulin have been implicated in cellularageing (Uppuluri et al. 1993). Both a- and b-tubulin havebeen shown to associate with the amyloid deposits of familialamyloidosis (Baumann et al. 1996) and to bind to the Absequence of APP (Islam and Levy 1997). Moreover,b-tubulin was retained by a monomeric Ab column (Oyamaet al. 2000). Our finding of an interaction between fAb andb-tubulin confirms the role of the latter protein in theaetiology of AD.

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The fact that synapsins co-precipitated with Ab may behighly significant in AD. Synapsin I and II are abundantsynaptic vesicle proteins, which belong to a family ofneurone-specific phosphoproteins. They are highly concen-trated in presynaptic nerve terminals, where they areassociated with the cytoplasmic surface of synaptic vesicles,playing a key role in neurotransmitter release and in theformation and maintenance of synaptic contacts amongcentral neurones (Ferreira and Rapoport 2002). It has beendemonstrated recently that there is regional loss of synapsin Iin the hippocampal formation of patients with late-stage AD(Qin et al. 2004). It has been suggested that the regionaldecrease in synapsin should be associated with cytoskeletalchanges as well as with Ab deposits, but not in the presenceof Ab plaques alone (Mukaetova-Ladinska et al. 2000; Qinet al. 2004). The present report is the first to suggest aninteraction between synapsins and fAb.

Co-precipitation of fAb 2¢,3¢-cyclic nucleotide 3¢-phos-phodiesterase 1 (CNPase1) indicates that Ab may contributeto the pathogenesis of AD by disturbing myelination.CNPase1 is a myelination-specific polypeptide, thought tohave a role in the synthesis and maintenance of myelinsheath (Yin et al. 1997). In a dog model of AD, myelinpathology was characterized by a loss of periodicity, adecrease in electron density and by membrane discontinuities(Torp et al. 2000). Moreover, biochemical analyses revealedincreased quantities of Ab1–40 and Ab1–42 in the whitematter of patients with AD, accompanied by significantdecreases in the amounts of myelin basic protein and CNPase1 (Vlkolinsky et al. 2001; Roher et al. 2002). In addition,fAb1–42 appeared to be continuous with the myelin sheathand to infiltrate axons through the myelin (Torp et al. 2000).The identification of myelin-associated molecular partners offAb might be of significance in AD.

Vacuolar proton-pump, ATP synthase subunit C(V-ATPase C) was also co-precipitated with fAb. V-ATPaseis a multisubunit enzyme that translocates protons across theplasma membrane and membranes of various organelles. Inneurones, the proton gradient generated by V-ATPase is usedby specific vesicular transporters to accumulate the neuro-transmitters in synaptic vesicles. V-ATPase is composed oftwo subcomplexes, V(1) and V(0). Hydrolysis of ATP in theV(1) subcomplex is tightly coupled to proton translocationaccomplished by the V(0) subcomplex, which is composedof five subunits (A, D, C, C¢, and C¢). Subunit c is a smallhighly hydrophobic integral membrane protein, necessary forthe assembly of the catalytic sector of the enzyme (Flanneryet al. 2004). In addition to this well established role,V-ATPase C has been shown to bind to the actin cytoske-leton, probably functioning as an anchor protein regulatingthe linkage between V-ATPase and the actin-based cytoske-leton (Vitavska et al. 2003). Possible perturbations of thesetwo important functions of V-ATPase C – participation inneurotransmitter storage and linking to the cytoskeleton – are

in accordance with the pathological processes describedfor AD.

In conclusion, our work shows that many abundantproteins co-precipitate with fAb, indicating strongly thatfAb might modulate (probably corrupt) the function of theseproteins. Further work is needed to differentiate betweenproteins precipitated by direct binding to fAb and otherproteins that are participants of a protein network. However,direct binding seems the most probable, as the majority of theidentified proteins have already been associated with AD.The fact that fAb interacts with such a wide variety ofproteins may provide an explanation for its divergent effects,which may vary depending on its concentration, aggregationstate and cell type.

In the future, the role of the binding process of Abaggregates to different proteins (co-aggregation) in theaetiology of AD should be investigated. Our experimentalmodel can be used to identify the proteins precipitated by otherfibrillar proteins involved in neurodegenerative disorders.

Acknowledgements

This work was supported by the National Council for Research and

Technology (NKFP) Budapest (RET 08/2004), and also by QLK1-

CT-2002-00172 EU-5 LIPIDIET and QLK1-CT-1999-02004 MA-

NAD grants. We would like to thank Ildiko Nemeth (Biological

Research Center, Szeged), Katalin Soos, Zsolt Datki (University of

Szeged, Szeged), Julianna Kardos and Eva Szarics (Chemical

Research Center, Budapest) for their kind help.

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