Binding of neural cell adhesion molecules (N-CAMs) to the cellular prion protein1

doi:10.1006/jmbi.2001.5183 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 314, 1209±1225

Binding of Neural Cell Adhesion Molecules (N-CAMs) to the Cellular Prion Protein Gerold Schmitt-Ulms1,2, Giuseppe Legname1,2, Michael A. Baldwin1,2,7 Haydn L. Ball1,2, Nicole Bradon1,2, Patrick J. Bosque1,2 Kathryn L. Crossin8, Gerald M. Edelman8, Stephen J. DeArmond1,5 Fred E. Cohen1,3,4,6 and Stanley B. Prusiner1,2,4* 1

Institute for Neurodegenerative Diseases 2

Department of Neurology

3 Department of Cellular and Molecular Pharmacology 4 Department of Biochemistry and Biophysics 5

Department of Pathology

6

Department of Medicine and

7

Department of Pharmaceutical Chemistry, University of California, San Francisco CA 94143, USA 8

Department of Neurobiology The Scripps Research Institute SBR-14, 10550 North Torrey Pines Road, La Jolla CA 92037, USA

To identify molecular interaction partners of the cellular prion protein (PrPC), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrPC. Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrPC in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identi®ed three murine splicevariants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAMspeci®c peptide library, the PrP-binding site was determined to comprise b-strands C and C0 within the two consecutive ®bronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144154) and the adjacent loop region of PrP. N-CAM-de®cient (N-CAMÿ/ÿ) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (4.1, SEM) days, arguing that N-CAM is not involved in PrPSc replication. Our ®ndings raise the possibility that N-CAM may join with PrPC in carrying out some as yet unidenti®ed physiologic cellular function. # 2001 Academic Press

*Corresponding author

Keywords: prion protein; protein X; formaldehyde crosslinking; cell adhesion; scrapie

Introduction Prion diseases are neurodegenerative disorders.1 In the past decade, public awareness of these diseases has grown in parallel with the increase in Abbreviations used: PrP, prion protein; PrPSc, scrapielike isoform of PrP; PrPC, cellular isoform of PrP; nvCJD, new variant Creutzfeldt-Jakob Disease; Hu, human; Mo, mouse; N2a, mouse neuroblastoma cell line; ScN2a, scrapie-infected mouse neuroblastoma cell line; N-CAM, neural cell adhesion molecule; Tg, transgenic; CLDs, caveolae-like domains; HMM-PrP complexes, high molecular mass PrP-containing complexes; FA, formaldehyde; GPI, glycosylphosphatidyl inositol; RML, Rocky Mountain Laboratory. 0022-2836/01/051209±17 $35.00/0

cases of bovine spongiform encephalopathy (BSE) and new variant Creutzfeldt-Jakob disease (nvCJD) in Europe. The normal cellular prion protein, denoted PrPC, undergoes a structural transition to its disease-causing isoform (PrPSc) with profoundly different physicochemical properties. Mutations in the PrP gene have been shown to cause inherited prion diseases in humans2 and transgenic (Tg) mice,3,4 both of which are transmissible to laboratory animals.5 ± 7 The conversion of PrPC to PrPSc seems to require localization of PrPC to lipid rafts,8 ± 12 which are specialized membrane regions rich in cholesterol and sphingolipids; however, the precise physiological environment in which this process occurs remains elusive. # 2001 Academic Press

1210 Although the cellular function of PrPC is unknown, its localization to the cell surface suggests involvement in processes related to cell adhesion, ligand uptake, recognition, or transmembrane signaling. In a cellular model of neuronal development, antibody-based crosslinking of PrPC induces phosphorylation of the tyrosine kinase Fyn in a manner consistent with a signaling cascade.13 As PrPC is glycosylphosphatidyl inositol (GPI)anchored and lacks a transmembrane segment, it is likely that a second protein participates with PrPC in signal transduction. The ability of PrPC to bind copper (II) ions through histidine residues within four glycine-rich octarepeats near its N terminus14 ± 19 would be consistent with a role of PrPC in copper metabolism. However, it has been well documented that PrP-ablated (Prnp0/0) mouse models that do not upregulate expression of the PrP-like protein doppel display no gross developmental or anatomical defects.20 In studies of the transmissibility of human PrPSc in wild-type and Tg mice, genetic evidence for the importance of a second host factor in PrPSc replication emerged.5 Additional studies mapped the binding site of this auxiliary factor, provisionally designated ``protein X'', to a discontinuous epitope consisting of four residues (Q168, Q172, T215 and Q219) on the surface of PrPC.21,22 In an effort to identify protein X, numerous candidate proteins that bind to PrP by two-hybrid (THS) or expression library screening experiments have been considered.23 ± 25 These molecular biological approaches share intrinsic limitations when studying membrane proteins or interactions of a transient or weak nature. To overcome these constraints, we sought to apply a biochemical approach that would preserve the integrity of the microenvironment of PrPC and covalently stabilize interactions of PrPC with its neighbors. Mild formaldehyde crosslinking has been used extensively for the study of nucleosomal protein interactions.26 ± 28 More recently, its usefulness for the study of membrane protein interactions has been recognized.29 Features that make formaldehyde crosslinking attractive are: (i) the water solubility of the reagent; (ii) the absence of reagentinduced re-arrangements of the proteins; and (iii) Ê ), endure the crosslink bonds are short (2 to 3 A harsh, non-physiological treatments and are reversible.26 Because controlled formaldehyde crosslinking on brain samples would require prior disruption of tissue integrity, we based our studies on mouse neuroblastoma (N2a) cells, an established cell model for the study of prion replication.30,31 We report that mild formaldehyde crosslinking of mouse N2a cells trapped a major fraction of PrPC in high molecular mass (HMM) protein complexes of 200 to 225 kDa. These complexes puri®ed with detergent-resistant glycolipid rafts, maintained integrity upon treatment with PNGase F and resisted release by phosphatidyl inositol phospholipase C (PIPLC). Puri®cation led to identi®-

PrP Interacts with N-CAM

cation of isoforms of the neural cell adhesion molecule (N-CAM) as components of these complexes. The N-CAM/PrP interface was characterized using an N-CAM-speci®c peptide library and truncated PrP constructs. We also found that NCAM was not required for PrPSc formation. Whether N-CAM joins with PrPC to carry out some as yet unidenti®ed cellular function remains to be established.

Results PrPC is a component of HMM complexes In search of proteins that interact with PrPC, our objective was to establish a crosslinking protocol that preserves the physiological environment of PrPC as authentically as possible. All biochemical studies were performed on mouse N2a cells and their Rocky Mountain Laboratory (RML) prioninfected derivatives (ScN2a). PrPSc from ScN2a cells display the characteristic partial resistance to treatment with proteinase K (PK) (Figure 1(a)). In situ crosslinking of N2a cells in the presence of >1 % formaldehyde resulted in the identi®cation of PrPC as a component of HMM complexes of 200 to 225 kDa (HMM-PrP complexes) (Figure 1(b), lanes 2-4 and 6-8). These complexes can be detected with various antibodies directed against PrP (shown for chimeric human-mouse (HuM) recombinant antibody fragment (Fab) D13). Increasing concentrations of formaldehyde caused an increase in signal intensity and the appearance of additional bands of primarily higher molecular mass (Figure 1, compare lanes 2, 3 and 4). Infection of the N2a cell clone with RML prions did not alter the band pattern of the HMM-PrP complexes, but caused a reduction in signal intensity (Figure 1, compare lanes 2-4 and 6-8). Previous work indicated that the conversion of PrPC to PrPSc takes place in caveolae-like domains (CLDs).11,12 CLDs can be puri®ed on sucrose gradients by exploiting their relative low density.8,32 To explore whether the HMM-PrP complexes and CLDs co-enrich, N2a and ScN2a cells were crosslinked in situ in the presence of 1 % formaldehyde. Upon extraction with cold Triton X-100 and ultracentrifugation, CLDs were harvested from the 5 %-30 % sucrose transition region of a sucrose step gradient. Additional fractions of interest were collected along the gradient and analyzed by immunoblotting with the HuM-D13 recombinant Fab directed against PrP. While uncrosslinked PrP gave rise to strong signals in both the pellet and CLD fraction of the sucrose gradient (Figure 2(a), lanes 1, 4, 5 and 8), the HMM-PrP complexes were almost exclusively found in the CLD fraction (Figure 2(a), lanes 4 and 8). Comparison of signal intensities of bands originating from uncrosslinked and crosslinked PrP within the CLD fractions revealed that a signi®cant proportion of the total PrP within this fraction is part of the HMM-PrP complexes (Figure 2(a), lanes 4 and 8).

1211

PrP Interacts with N-CAM

Figure 1. PrP is a component of HMM complexes. (a) Mouse N2a cells before (lanes 1, 2, 5, 6) or after infection with RML prions (ScN2a) (lanes 3, 4, 7, 8) were grown to con¯uency. Crude cellular extracts were either left untreated (lanes 1-4) or subject to digestion with PK (lanes 5 and 7: 10 mg/ml PK; lanes 6 and 8: 20 mg/ml PK) for one hour at 37  C. Samples were analyzed by immunoblotting using the Fab HuM-D13. (b) Mouse N2a cells before (lanes 1 to 4) or after infection with RML prions (lanes 5 to 8) were left untreated (lanes 1 and 5) or crosslinked in situ with the formaldehyde concentration shown, for 15 minutes at RT (lanes 2-4 and 68). Immunoblot detection of PrP was performed from crude cellular extracts using the Fab HuM-D13.

To address whether N-linked sugar molecules are directly involved in the crosslinking reaction, we took the crosslinked N2a and ScN2a cells, harvested their cellular extracts, and digested them enzymatically with PNGase F. Treatment with PNGase F signi®cantly increased the mobility of the HMM-PrP complexes, but did not break up the complexes into their components (Figure 2(b), lanes 4 and 8). While N2a and ScN2a cells shared this behavior, signal intensities for the crosslinked

products were again somewhat lower in the permanently infected cell line (Figure 2(b)), compare lanes 4 and 8). Treatment with PIPLC releases PrPC from the cell surface through cleavage of its glycosylphosphatidyl inositol (GPI) anchor.33 To explore whether HMM-PrP complexes are bound to the membrane by means other than GPI-mediated membrane attachment, cells were subjected to treatment with PIPLC prior to or after crosslinking in the presence of 5 % formaldehyde (Figure 2(c)). When PIPLC treatment preceded the crosslinking reaction, it prevented formation of HMM-PrP complexes (Figure 2(c), lanes 4 and 10) and caused the release of uncrosslinked PrPC migrating at 20 to 35 kDa under all conditions tested (Figure 2(c), even numbered lanes). When PIPLC treatment followed crosslinking, it depleted cells of the uncrosslinked fraction of PrP, but was no longer able to release PrP participating in HMM-PrP complexes that migrate at 200 to 225 kDa (Figure 2(c), lanes 6 and 12). Taken together, it appears that the PrP component of the HMM-PrP complex is recruited from a pool of PIPLC-sensitive PrP. Once the HMM-PrP complexes are formed, they become PIPLC-resistant and therefore must employ a mode of membrane attachment that does not rely solely on the GPI anchor provided by PrP itself. HMM-PrP complexes were puri®ed to homogeneity in three steps. First, N2a cells were crosslinked in situ in the presence of 1 % formaldehyde and cell extracts were loaded onto a Q-Sepharose anion exchange column. While the majority of uncrosslinked PrPC was found in the unbound fraction (Figure 3(a), lane U), the HMM-PrP complexes were quantitatively retained on the ion exchange matrix, subsequently eluted in the presence of >200 mM sodium chloride (Figure 3, eluate fractions), and then immunoaf®nity puri®ed using biotinylated recombinant Fabs HuM-D13 and HuMD18 pre-bound to streptavidin agarose. As shown in Figure 3(b), a pre-clearance step (without Fabs) employed to remove nonspeci®c binders to the streptavidin beads failed to bind any PrP-containing complexes. In contrast, in the presence of Fabs, the use of the biotin/streptavidin system was compatible with high stringency washes of the loaded beads and proved to quantitatively bind the HMM-PrP complexes (Figure 3(b)). A ®nal separation of the HMM-PrP complexes on a preparative sodium dodecyl sulfate-6 % polyacrylamide gel electrophoresis (SDS-PAGE) removed residual uncrosslinked PrPC and traces of antibodies and streptavidin. N-CAM is a major component of the HMM-PrP complexes To determine the molecular identity of any nonPrP components of the HMM-PrP complexes, the relevant HMM bands were excised and in-gel trypsinized. Extracted peptides were analyzed using electrospray tandem mass spectrometry. Peptide

1212

PrP Interacts with N-CAM

Figure 2. Biochemical characterization of HMM-PrP complexes. HMM-PrP complexes (a) co-isolate with CLDs, (b) maintain integrity following removal of N-linked sugar moieties and (c) resist release by PIPLC. (a)-(c) Studies were carried out with mouse N2a cells before or after infection with RML prions (ScN2a). Samples were analyzed by immunoblotting employing the Fab HuM-D13 to detect PrP. (a) Cells were crosslinked in situ in the presence of 5 % formaldehyde (15 minutes at RT). Cold Triton X-100 extracts were loaded onto a sucrose step gradient (gradient steps from bottom to top: 80 %, 40 %, 30 %, 5 % sucrose). Critical fractions along the gradient, including the 30 %-5 % transition fraction enriched in CLDs, were collected from the gradient transition regions and N2a cells were analyzed by immunoblotting. (b) Cells were either left untreated (lanes 1, 2, 5, 6) or crosslinked in situ in the presence of 5 % formaldehyde (15 minutes at RT, lanes 3, 4, 7, 8). Subsequently, cellular extracts were split into duplicates that were either subjected to enzymatic digestion with PNGase F (1.0 unit/20 mg protein, 16 hours, 30  C; lanes 2, 4, 6, 8) or left undigested (lanes 1, 3, 5, 7). Equal aliquots of protein were analyzed via immunoblotting. (c) Cells were either left untreated (lanes 1, 2, 7, 8) or crosslinked in situ in the presence of 5 % formaldehyde (15 minutes at RT; lanes 3-6, 912). In situ digestion with PIPLC was performed on control extracts (lanes 2, 8) and before (lanes 4, 10) or after (lanes 6, 12) the crosslink reaction. Equal aliquots of protein extracts were analyzed via immunoblotting.

mass searches of the NR-database (NCBI) indicated the presence of mouse N-CAM splice-variants in both the 200 kDa and 225 kDa HMM-PrP com-

plexes. Fragment ion data were collected for a total of 18 peptides. Computational and manual collision-induced dissociation (CID) spectra interpret-

PrP Interacts with N-CAM

1213

Figure 3. Isolation of HMM-PrP complexes in three steps. Upon crosslinking in the presence of 1 % formaldehyde (a) N2a cell extracts were passed over Q-Sepharose HP anion exchange medium and HMM-PrP complexes were eluted with a 100 mM to 600 mM linear salt gradient. (b) Pooled eluates were pre-cleared using bovine serum albumin (BSA)-blocked streptavidin agarose beads (ÿFabs). Subsequently, unbound protein was subjected to immunoaf®nity chromatography employing streptavidin agarose beads that had been pre-saturated with Fabs and blocked with BSA (‡Fabs). Control aliquots from (a) and (b) were separated on 4 %-12 % denaturing gels and analyzed via immunoblotting using the Fab HuM-D13. L, sample load; U, unbound material; W, wash; E, eluate fractions.

ation allowed meaningful protein assignment for the ®ve of these data sets. In agreement with the initial assignment, the ®ve strongest data sets returned isoforms of N-CAM as the best hits. In one instance, ten out of ten fragment ion masses matched predicted N-CAM fragment masses, con®rming the presence of N-CAM in the HMM complexes (Table 1). Different N-CAM splice-variants found in HMM-PrP complexes Alternative splicing in mice and humans generates three predominant N-CAM isoforms,34 two of which are transmembrane (N-CAM-180 and NCAM-140) and a shorter isoform which is GPIanchored (N-CAM-120) and localizes to lipid rafts.35 To verify the presence of N-CAM in the HMM-PrP complexes and to determine the contribution of the different splice variants to the 200 kDa and 225 kDa PrP-containing complexes, immunoblots were produced using antibodies directed against either PrP (Figure 4(a)) or the extracellular domain of N-CAM that is common to all three major isoforms of N-CAM (Figure 4(b)). Samples for this experiment were either extracts from a formaldehyde-crosslinked concentration series (Figure 4(a) and (b), lanes 1 to 4) or HMMPrP complexes (Figure 4(a) and (b), lanes 5 to 8) puri®ed according to the three-step puri®cation

scheme outlined above (Figure 3). The latter were loaded either untreated or after boiling for various durations in the presence of an excess of NH2groups (provided by Tris), a treatment that has been demonstrated to revert formaldehyde-based crosslinking.26 Upon prolonged heat treatment, HMM-PrP complexes disappeared, as demonstrated in the crosslink reversal time series (Figure 4(a) and (b), lanes 5 to 8). When the immunoblot was reprobed with an N-CAM-speci®c antibody, HMM-PrP complexes were not readily detected in the extract fractions (Figure 4(b), lanes 1 to 4). Instead, uncrosslinked N-CAM was found to migrate with apparent molecular masses of 200, 175 and 165 kDa (Figure 4(b), lane 1) and formed crosslinked bands in the range of 200 to 300 kDa (Figure 4(b), lanes 2 to 4). Only with puri®ed HMM-PrP complexes did it become apparent that the N-CAM-speci®c antibody detected the 225 and 200 kDa bands with relative signal intensities that matched those observed with the PrP-directed antibody (Figure 4(b), lane 5). During the course of the heat treatment, HMM-PrP complexes were gradually replaced by bands that migrated with increased mobility (corresponding to a difference in apparent molecular mass of about 25 kDa: Figure 4(b), lanes 6 to 8), giving rise to signals that in terms of their molecular mass and relative intensity migrated with uncrosslinked splice isoforms of N-CAM (Figure 4(b), lane 1).

Table 1. Mass spectrometrical identi®cation of N-CAM splice-variants in HMM-PrP complexes HMM-PrP complexes (kDa) Data type LC/MS

LC/MS/MS

a

200 ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡

225 ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡

‡

Monoisotopic massesa MH‡ Submitted

Matched

1031.56 707.40 1648.96 2025.01 1168.61 747.48 1535.80 1222.68 1377.68 1183.64 847.50 1300.72 2334.22 2668.48 1168.61 1222.64 1393.66 1199.62 2668.48

1031.51 707.37 1648.93 2024.92 1168.59 747.46 1535.77 1222.63 1377.63 1183.61 847.45 1300.67 2334.14 2668.39 1168.59 1222.64 1393.62 1199.61 2668.39

For LC/MS/MS data, parent masses given only. b The only N-CAM-140 speci®c peptide found in 200 kDa HMM-PrP complex. c The only N-CAM-180 speci®c peptide found in 225 kDa HMM-PrP complex.

N-CAM sequence (Acc. #: P13595) Tryptic peptide (single AA code) (K)NAPTPQEF(K) (R)GEINFK(D) (K)DIQVIVNVPPTVQAR(Q) (R)NVDKNDEAEYVCIAENK(A) (K)AGEQDASIHLK(V) (R)VSSLTLK(S) (R)DGQLLPSSNYSNIK(I) (K)GLGEISAATEFK(T) (K)LEGQMGEDGNSIK(V) (R)LPSGSDHVMLK(S) (K)AAHFVFR(T) (K)DESKEPIVEVR(T) (K)HTEPNETTKGPVETK(S)b (K)ASPAPTPTPAGAASPLAAVAAPATDAPQAK(Q)c (K)AGEQDASIHLK(V) (K)GLGEISAATEFK(T) (K)LEGQMGEDGNSIK(V) ‡ Met-ox (R)LPSGSDHVMLK(S) ‡ Met-ox (K)ASPAPTPTPAGAASPLAAVAAPATDAPQAK(Q)

203

Start

End

122 197

130 202 217 293 304 374 455 595 619 662 691 782 811 974 304 595 619 662 974

277 294 368 442 584 607 652 685 772 796 945 294 584 607 652 945

1215

PrP Interacts with N-CAM

M

Taken together, these ®ndings argue for the presence of all three major isoforms of N-CAM in HMM-PrP complexes. Quantitative contributions of N-CAM isoforms to the formation of HMM-PrP complexes seem to re¯ect their relative abundance in the cell with the 140 kDa isoform being most predominant. The molecular masses of the HMMPrP complexes and their components as well as the gel mobility shift observed upon crosslink reversal are consistent with a PrP to N-CAM stoichiometry of 1:1. Recombinant PrP binds to FNIII-like domains of N-CAM To characterize the interface between N-CAM and PrP, a multi-pin peptide library was generated. N-CAM-speci®c peptides were synthesized onto an array of polypropylene pins using 9-¯uorenylmethoxycarbonyl (Fmoc) chemistry. Additional array peptides were synthesized that served as either negative or positive controls (see the legend to Figure 5). Detection of binding followed a standard ELISA procedure. When carried out without prior incubation with recombinant PrP (rPrP), this procedure itself gave rise to comparably weak signals at a small number of pin positions. Under these test run conditions, a strong signal was only obtained for one peptide per plate that served as a positive control, con®rming the success of the library synthesis and the speci®city of the detection system employed (Figure 5(a), upper panel). The library was then stripped and reprobed with full-length mouse rPrP that had been folded into its predominantly a-helical conformation (as con®rmed by CD spectroscopy, data not shown). The subsequent detection of binding events con®rmed the propensity of PrP to adhere to surfaces. Under these conditions, a background of weak signals was obtained for about half the pin positions. More importantly, absorbance values for three clusters of ®ve to seven consecutive peptides each were as high as absorbance values obtained from the internal positive controls (Figure 5(a), lower panel). Conversely, highly acidic, basic, polar, and hydrophobic non-N-CAM array peptides that were included as negative controls gave rise to absorbance values of little above background. Quantitative analysis con®rmed this initial assignment and identi®ed continuous stretches of sequence from within the Ig-5 and the two FNIII-like domains of N-CAM as the putative rPrP binding sites (Figure 5(b)). It is well established that Ig-like

Figure 4. Immunoblotting con®rms interaction of PrP with all three major isoforms of N-CAM. Mouse N2a cells were left untreated (lane 1), crosslinked in situ in the presence of 0.5 to 2.5 % formaldehyde (lanes 2 to 4), or crosslinked in the presence of 1 % formaldehyde, puri®ed according to three-step protocol and subjected to crosslink reversal conditions for different durations (0 second, 90 seconds, ®ve minutes or 30 minutes)

(lanes 5-8). Identical samples ((a) and (b)) were subjected to 4 %-12 % SDS-PAGE and immunoblotting using (a) the PrP-directed Fab HuM-D13 or (b) a monoclonal antibody (mAb) directed against all three major isoforms of N-CAM. (c) Explanatory diagram showing the position of the three N-CAM-PrP complexes in (b), lane 5.

1216

PrP Interacts with N-CAM

Figure 5 (legend opposite)

1217

PrP Interacts with N-CAM

domains and FNIII domains share signi®cant sequence homology and adopt a highly similar fold.36 Figure 5(d) demonstrates that all three NCAM regions contributing strongest to PrP binding (N-CAM library peptides 81-83, 102-107 and 124128) map to predicted strands C and C0 within a multiple alignment of N-CAM domains. While no structural information is available for the FNIII domains of N-CAM, the structure of the third FNIII module of tenascin has been solved and found to be topologically identical with the C2-set of Ig domains (i.e. a b-sandwich made up of a four-stranded and a three-stranded antiparallel b-sheet consisting of strands G-F-C-C0 and A-B-E, respectively). We used this third FNIII domain of tenascin as a template to model the FNIII-1 domain of N-CAM (Figure 5(c)). According to this structure prediction, strands C and C0 form a continuous epitope. Segments of sequence from within these strands that contribute strongest to PrP binding are solvent exposed. Truncation of PrP compromises its ability to form HMM-PrP complexes To explore which segment(s) within PrP participate in the N-CAM/PrP interface, we studied the formaldehyde crosslink behavior of truncated PrP constructs. N2a cells stably expressing chimeric mouse-hamster (MHM2) constructs of PrP (carrying the 3F4 epitope) were crosslinked in situ in the

presence of 5 % formaldehyde. Cellular extracts were analyzed via immunoblotting using either the 3F4 antibody that does not cross-react with mouse PrP and therefore only recognizes the MHM2 PrP expression products (Figure 6(a)) or the HuM-D13 Fab that detects total PrP (Figure 6(b)). As expected, full-length MHM2 predominantly formed HMM-PrP complexes migrating at 200 to 225 kDa (Figure 6(a), lane 1). Two of the N-terminally truncated MHM2 constructs (
23-88;
2388,
122-140) gave rise to the appearance of crosslink bands of 50 to 60 kDa, suggesting the interaction with additional proteins and caused a signi®cant reduction in signal intensity for the bands of HMM-PrP complexes (Figure 6(a), lanes 2 and 3). Interestingly, HMM-PrP complexes disappeared when we employed PrP106 (
23-88,
141176; Figure 6(a), lane 4). Our ®ndings suggest a complex binding epitope on the PrP-side of the PrP/N-CAM interface; this epitope comprises residues from the N terminus, helix A and the adjacent loop region of PrP. N-CAM-deficient mice are susceptible to challenge with prions To test whether N-CAM de®ciency renders mice resistant to the infection by prions, N-CAM de®cient (N-CAMÿ/ÿ) mice and CD-1 control mice were intracerebrally inoculated with RML prions; uninoculated animals served as negative controls.

Figure 5. PrP binds to b-strands C and C0 within FNIII-like domains of N-CAM. (a) The N-CAM-speci®c multi-pin peptide library consisted of 140 different peptides, each 15 amino acid residues long and covalently attached to a pin through its C terminus. Consecutive peptides were shifted along the primary structure of N-CAM by ®ve amino acids and therefore displayed a ten amino acid residue sequence overlap. This arrangement gave rise to a library that covered the extracellular domain of mouse N-CAM (Acc. #: P13594) with a threefold redundancy. As indicated by position numbers, library peptides were arranged in order onto an array of two 96-pin blocks. Additional peptides were synthesized to serve as positive (pin positions indicated by the letter c: epitope from within PrP recognized by the HuM-D13 antibody) or negative (pin positions 143 and 144: random non-N-CAM; pin positions 145 to 148: highly acidic, basic, polar, and hydrophobic non-N-CAM peptides, respectively) controls. Pin positions indicated by the letter u were kept unmodi®ed. The peptide library was left untreated (upper panel) or incubated with full-length mouse rPrP (lower panel). In both instances, detection of binding events followed a standard ELISA procedure that was based on the HuM-D13 Fab, a peroxidase-labeled secondary antibody and the peroxidase substrate QuantaBlu (Pierce). (b) Quantitative analysis of multi-pin capture ELISA results following a four-step procedure. First, plate-toplate differences of the two 12  8 array plates that together make up the library were normalized based on the signal intensities provided by the identical positive control peptides found on each plate (pin positions labeled c). Given the threefold redundancy of the library, the binding contribution of a given ®ve amino acid residue segment could then be calculated by adding the absorbance values of the three consecutive pins that contain its sequence. After subtraction of the background signals (provided by pin positions labeled u), these binding contributions were plotted against the primary structure of N-CAM. Values are given as a percentage of the ELISA response (3) of the positive control peptides (pin positions labeled c). The schematic drawing depicting the domain structure of the extracellular domain of N-CAM is to scale. (c) Depiction of the rPrP binding site in a structure prediction of the FNIII-1 domain of N-CAM. The structure of the FNIII-1 domain of N-CAM was modeled based on atomic coordinates available from a crystal structure of the FNIII-3 domain of tenascin (PDB Id: 1TEN68). The Figure was generated with MOLSCRIPT69 and rendered with Raster3D.70 Side-chains and backbone of strong (>90 %), intermediate (60-90 %) and weak (30-60 %) binding segments are depicted in red, orange and yellow, respectively. Segments of the FNIII-1 domain that displayed very weak binding (10-30 %) are shown with a green backbone trace; blue represents no binding (<10 %). (Percentage values for the color assignment were derived from panel b). (d) Identi®cation of rPrP binding segments (N-CAM library peptides 81-83, 102-107 and 124-128) in multiple alignment of Ig-5 and FNIII domains of N-CAM. Note that rPrP binds to N-CAM predominantly within sequences corresponding to b-strands C and C0 of homologous N-CAM domains. Residues for which the alignment revealed identity, strong and weak similarity are indicated with different shades of green.

1218

PrP Interacts with N-CAM

All N-CAMÿ/ÿ mice inoculated with RML prions lived long enough to develop disease, with clinical signs of scrapie after 110 to 133 days (Table 2). As expected, uninoculated N-CAMÿ/ÿ mice did not develop signs of disease. To con®rm the clinical diagnosis of prion disease, inoculated mice with CNS dysfunction and controls were sacri®ced and brain extracts were examined for PrPSc by immunoblotting (Figure 7). Band smearing, the presence of partially degraded PrP (Figure 7(a), lanes 2 to 4) and PK-resistant PrP (Figure 7(b), lanes 2 to 4), characteristic of the presence of PrPSc, were detected in all RML-inoculated N-CAMÿ/ÿ mice, but were absent in brain extracts of uninoculated mice (Figure 7(a) and (b), lanes 5 to 7). Pathologic examination of coronal brain sections of inoculated N-CAMÿ/ÿ mice showing symptoms of CNS dysfunction revealed widespread PrPSc deposition and astrocytic gliosis typical for experimental scrapie (data not shown). Taken together, these data establish that the presence of N-CAM is not required for the replication of PrPSc.

Discussion

Figure 6. PrP binds to N-CAM through a discontinuous binding site comprising its N terminus, segments from helix A and the adjacent loop region. N2a cells were stably transfected with a series of MHM2 PrP expression plasmids that encode for truncated and/or internally deleted PrP constructs. Stable transfectants were crosslinked in situ with formaldehyde and extracts were subject to immunoblotting employing (a) the PrPdirected mAb 3F4 that does not cross-react with mouse PrP and therefore only recognizes the MHM2 PrP expression products or (b) the Fab HuM-D13 that detects total PrP. Please note that expression levels of all constructs were similar. Levels for the MHM2 PrP106 (
23-88,
141-176) construct appear lower because the protein lacks N-linked carbohydrates, which causes it to migrate as a single slim band in the SDS-PAGE.

In the studies reported here, we employed in situ crosslinking of N2a cells as an approach to identify PrPC-interacting proteins. We demonstrated that a signi®cant proportion of membrane-bound PrPC is entrapped in HMM complexes and showed that these complexes isolate with lipid rafts, maintain integrity upon treatment with PNGase F and resist release by PIPLC. Tandem mass spectrometry and immunoblotting identi®ed the non-PrPC component of these complexes to be isoforms of N-CAM. The PrP/N-CAM interface was characterized with the use of an N-CAM-speci®c peptide library and truncated PrP expression constructs. N-CAMÿ/ÿ mice challenged with RML prions succumbed to disease after a mean incubation period of 120 days. The spatial organization of GPI-anchored proteins such as PrP remains poorly understood. While some studies support the idea of a dispersed homogenous distribution,32,37 other evidence favors a dense packing of GPI-anchored proteins in specialized membrane regions rich in cholesterol and sphingolipids commonly referred to as CLDs.38 ± 41 Central to this controversy has been the question of whether detergent insoluble, glycolipid-enriched vesicles (DIGs) that can be isolated by exploiting their buoyancy on sucrose gradients42 represent artifacts of solubilization or discrete patches of cell-surface membrane. If CLDs exist, are they homogenous in the sense that all proteins within this space have access to each other? Alternatively, are there structurally and functionally specialized regions within CLDs that harbor different sets of GPI-anchored proteins at different densities?43 Insights into the spatial organization of GPIanchored proteins are likely to help us understand

1219

PrP Interacts with N-CAM Table 2. Inoculation of N-CAM-de®cient mice with RML prions Hosta N-CAMÿ/ÿ N-CAMÿ/ÿ CD-1 CD-1

Inoculum

n/n0b

Incubation time (days) (Mean  SEM)

RML RML -

8c/8 0/8 8/8 0/8

122  4.1 >200 128  2.9 >200

a N-CAMÿ/ÿ mice were characterized by an insertion of the bacterial lacZ gene between the transcription and translation initiation sites of the N-CAM gene. b n, number of scrapie-sick animals; n0, total number of animals inoculated. c Two mice of this group died of unrelated causes early after inoculation.

more about PrP biology and pathology. Both the cellular form and the scrapie conformer of PrP have been shown to purify with DIGs.9,11 Depleting cells of cholesterol caused a signi®cant decrease in PrPSc replication and slowed the rate of PrPC degradation.10,44 Redirecting PrPC by replacing its GPI-anchor sequence with transmembrane sequences that target PrPC to clathrin-coated pits prevented PrPSc replication in ScN2a cells.12 In the studies reported here, we applied formaldehyde crosslinking to study the PrP microenvironment in the cell. It is interesting that mild formaldehyde crosslinking (41 % formaldehyde, 15 minutes, RT) did not generate a complex band pattern, but instead caused the appearance of one distinct, HMM-PrP-containing band of 200 kDa. This suggests a relatively speci®c interaction. Additional bands of predominantly higher molecular mass only appeared in the presence of higher concentrations (52.5 %) of formaldehyde. When we analyzed the cellular distribution of these complexes, we observed a strong enrichment of the HMM-PrP complexes in low sucrose fractions after extraction with cold Triton X-100 and gradient centrifugation. Comparing the relative strengths of the bands originating from HMM-PrP complexes (when probed with a PrP-directed antibody) versus signals originating from uncrosslinked PrPC in the CLD fraction, it appears that about half of the PrP that had been targeted to CLDs was trapped in HMM complexes, suggesting that these complexes might serve some distinct function in the lifecycle of PrP. Mammalian PrPC contains two consensus sites for N-linked glycosylation, giving rise to un-, mono-, and diglycosylated forms of the protein. The presence of bi-, tri-, and tetra-antennary complex-type oligosaccharide chains, with a majority of the terminal galactose residues linked to sialic acid, has been reported.45 ± 47 A detailed comparison of PrPC- and PrPSc-linked sugars revealed that PrPSc contains increased levels of tri- and tetraantennary sugars and decreased levels of glycans with bisecting GlcNAc residues.48 To investigate whether the components within the HMM-PrP complexes are held together through interactions of carbohydrate moieties or represent bona ®de protein interactions, we subjected the complexes to treatment with PNGase F and observed the antici-

pated shift in molecular mass without disintegration of the complex into its components. While this observation does not rule out a participation of sugar molecules in the formation of the complex, it shows that the complex components are not merely held together by carbohydrate moieties. The HMM-PrP complexes were puri®ed employing a strategy that relied on the af®nity and speci®city of a mixture of biotinylated Fabs recognizing non-overlapping epitopes on PrP. Upon in-gel trypsinization, LC/MS/MS analysis allowed unambiguous identi®cation of N-CAM isoforms as non-PrPC components of the HMM-PrP complexes, a ®nding that subsequently was corroborated by Western immunoblot data with N-CAM-directed antibodies. Alternative splicing predominantly generates three isoforms of N-CAM, two transmembrane isoforms, generally referred to as N-CAM-180 and N-CAM-140, and one GPIanchored isoform known as N-CAM-120.34 Due to complex N-linked sugar and polysialic acid modi®cations, these isoforms migrate with apparent molecular masses of 200, 175 and 165 kDa, respectively. Assuming an apparent molecular mass of about 25 kDa for PrP, the apparent sizes of the 225 and 200 kDa HMM-PrP complexes are consistent with a 1:1 stoichiometry between PrP and N-CAM isoforms. A 1:1 stoichiometry would also explain why heat treatment and release of PrPC are paralleled by an approximately 25 kDa molecular mass shift (Figure 4(b), lanes 5 to 8). The existence of additional splice events limited to the extracellular domain of N-CAM and subjected to developmental and/or tissue-speci®c regulation has been reported.49 ± 51 Our current set of data cannot distinguish between these minor N-CAM subspecies. It appears that PrPC associates with all three major splice variants of N-CAM. Quantitative contributions of N-CAM isoforms to the formation of HMM-PrP complexes seem to re¯ect their relative abundance in the cell (N-CAM-140 isoform > N-CAM-180 isoform >> N-CAM-120 isoform). Stoichiometric binding of PrP to all three major isoforms of N-CAM requires that the association occurs at a time when PrPC has access to these isoforms. Given the likely non-homogenous distribution of N-CAM within the plasma membrane, an early association of PrPC with splice variants of N-CAM during their joint passage along the

1220

Figure 7. N-CAM-de®cient mice succumb to disease after intracerebral inoculation with RML prions. All inoculated N-CAMÿ/ÿ mice developed scrapie symptoms after 105 to 130 days. Brain extracts of scrapie-sick N-CAMÿ/ÿ mice (lanes 2 to 4), non-inoculated NCAMÿ/ÿ mice (lanes 5 to 7), and a C57Bl6 control mouse (lane 1) were analyzed via immunoblotting employing ((a), (b)) the PrP-directed HuM-D13 Fab or (c) the N-CAM-directed antibody RDI-N-CAM13abm. Extracts were either (a) left untreated or (b) subjected to digestion with PK (20 mg/ml) for one hour at 37  C.

PrP Interacts with N-CAM

secretory pathway is the most plausible scenario. Whereas PrPC is a low abundance protein, N-CAM represents a class of cell adhesion molecules that is expressed in moderately large quantities. We postulate that a signi®cant proportion of PrPC binds N-CAM but only a small fraction of the cellular N-CAM engages in interactions with PrPC. This hypothesis also accounts for the ability of N-CAM to engage in homophilic as well as heterophilic interactions with other proteoglycans, both between cells and within the same cell.52 ± 54 Employing a peptide library covering the extracellular domain of N-CAM, we identi®ed b-strands C and C0 within the two consecutive FNIII domains of N-CAM as candidate regions for binding to PrPC. Because short segments of sequence lacking the context of the surrounding protein molecule might adopt a non-physiological fold, caution needs to be applied when interpreting data of this kind. Despite these limitations, peptide libraries have proven valuable for the interface mapping of a large number of protein interactions and in particular, of antibody-binding sites. Three observations support the identi®cation of the FNIII domains of N-CAM as PrP binding sites: (i) the binding sites identi®ed do not represent peptides that stand out in terms of charge or hydrophobicity; (ii) the strongest binding occurred in three clusters of consecutive pins that represent continuous stretches of sequence with considerable homology; (iii) the model structure of the FNIIIdomain of N-CAM predicts that stretches of sequence contributing most strongly to PrP binding are solvent-accessible. Additionally, in light of the mobility constraints that are imposed on membrane-attached molecules, it makes sense that NCAM, as the much larger of the two binding partners, would employ membrane-adjacent FNIII domains for its binding to PrP. Interestingly, strands C and C0 of FNIII domains are structurally and phylogenetically related to the C and C0 strands of Ig-like variable domains that strongly contribute to antigen recognition. It remains to be seen whether binding to PrP under physiological conditions involves one or more of the three homologous N-CAM binding sites identi®ed in this study. Clearly, N-CAM is not the postulated auxiliary factor ``protein X'' implicated in PrPC conversion. The observation that N-CAMÿ/ÿ mice succumb to prion disease rules out that N-CAM is required for PrPSc formation. The incubation time study presented here employed a small number of animals that had not been back-crossed frequently enough to establish a pure inbred background. It therefore remains to be seen whether the small difference in incubation time of N-CAMÿ/ÿ mice and CD-1 mice observed here is signi®cant and re¯ects a modifying effect of N-CAM on incubation time. Interestingly, the mouse N-CAM gene maps to a quantitative trait locus on chromosome 9, postulated to encode a modi®er gene for prion incubation time.55

1221

PrP Interacts with N-CAM

Recently, it has been demonstrated that PrPC can function in a signal transduction cascade upstream of the protein tyrosine kinase Fyn.13 In that study, a neuroectodermal progenitor cell line (1C11) with an epithelial morphology56 was used to search for PrPC-dependent signal transduction through antibody-mediated crosslinking. PrPC-dependent Fyn activation was observed when the 1C11 cells had been differentiated to their serotonergic or noradrenergic progenies. Because PrPC and Fyn are bound to opposing faces of the membrane, a search for a transmembrane factor that could function as a link between PrPC and Fyn was initiated. Caveolin-1 was presented as a candidate based on its coimmunoprecipitative behavior and the observation that cellular bombardment with caveolin-1directed antibodies was shown to abolish PrPCdependent Fyn activation. Some cell lines, such as the N2a cell line used in this study, do not express caveolin,8,10 suggesting the existence of an alternative signaling route from PrPC to Fyn. Given the PrPC/N-CAM association evidenced here, it is tempting to speculate that such an alternative signaling cascade might involve N-CAM. This scenario gains support from an earlier set of data demonstrating the direct interaction of N-CAM and Fyn35,57,58 and the selective inhibition of N-CAM-dependent neurite outgrowth in neurons derived from fynÿ/ÿ mice.57 The identi®cation of N-CAM as a candidate for binding to PrPC opens new avenues of research and further characterization of this interaction will be of interest in terms of elucidating the cellular function of PrPC. It remains to be seen whether the ability of PrP to bind to FNIII domains within N-CAM extends to other proteins that contain this motif, such as L1. Future work will also be needed to clarify whether N-CAM acts as a modi®er of prion incubation time and facilitates peripheral accumulation of prions. Finally, the use of N-CAMÿ/ÿ mice might prove to be a valuable tool for the identi®cation of other PrP-interacting molecules whose binding is masked by the relative abundance of N-CAM.

Materials and Methods Antibodies Recombinant Fabs against PrPC were selected from phage display libraries prepared from Prnp0/0 mice immunized with infectious prions. These antibodies are expressed as HuM Fabs59 and detect non-overlapping epitopes (HuM-D13: SHaPrP amino acids 96-105, HuMD18: SHaPrP amino acids 133-157) on mouse PrP.60 AntiPrP 3F4 is a mAb raised against SHaPrP 27-30.61 RDI-NCAM13abm is a monoclonal mouse IgG2a antibody raised against mouse brain membrane fractions that recognizes the three major isoforms of N-CAM (Research Diagnostics, Inc., Flanders, NJ).

Recombinant prion protein Recombinant full-length mouse PrP was expressed from the pET11a plasmid in Escherichia coli (BL21DE3) in minimal media containing 100 mg/ml ampicillin. The bacterial paste was resuspended in 25 mM Tris-HCl, 5 mM EDTA (pH 8.0) and processed twice in a Micro¯uidizer M-110 EH (Micro¯uidics Corp., Newton, MA). Inclusion bodies were collected by centrifugation and solubilized in ®ve volumes of 8 M urea, 10 mM Mops (pH 7.0) by stirring overnight at RT. The puri®cation was based on successive chromatography on carboxy methyl sepharose and C4 reversed-phase media. rPrP adapted a predominantly a-helical conformation and was obtained 595 % pure as assessed by CD spectroscopy and mass spectrometry, respectively. Cell culture Mouse N2a were obtained from the American Tissue Culture Collection. ScN2a cells are clones persistently infected with the RML prion strain as described.30 Cells were grown and maintained at 37  C in minimal essential medium supplemented with 10 % fetal bovine serum and 1 % glutamax. MHM2 constructs for the stable transfection of N2a cells were as described62 and were maintained by the addition of 1 mg/ml geneticin to the cell culture medium. Proteinase K treatment Con¯uent cells were lysed in 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5 % NP-40, 0.5 % deoxycholate (DOC). Equal amounts of protein (determined using the bicinchoninic acid (BCA) reagent) were digested with 20 mg/ml PK (GIBCO, Carlsbad, CA) at a ratio of 1:25 (w/w), protease to protein, for one hour at 37  C. Reactions were stopped by the addition of 2 mM phenylmethylsulfonyl ¯uoride (PMSF). Following ultracentrifugation, pellets were resuspended in SDS-loading buffer and subjected to immunoblot analysis. Formaldehyde crosslinking and crosslink reversal All steps were carried out at RT unless noted. Cells were washed twice with phosphate buffered saline (PBS), pH 7.2. Crosslinking occurred during a 15-minute incubation in the presence of 1 to 5 % (w/v) formaldehyde in PBS, pH 7.2. Fixative was removed and the crosslink reaction was quenched with 125 mM glycine in PBS, pH 7.2 for ten minutes. Cells were washed twice with PBS and proteins were extracted from crosslinked cells with ice-cold extraction buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5 % NP-40, 0.5 % DOC). Equal amounts of protein (determined using the BCA reagent) were precipitated with acetone. Protein pellets were resuspended in either SDS-loading buffer without subsequent boiling or in crosslink reversal buffer (2 % SDS, 500 mM 2-mercaptoethanol, 250 mM Tris, pH 6.8) and heated for 1 to 30 minutes at 95  C.26 In both instances, samples were analyzed via immunoblotting. Enzymatic treatment of HMM-PrP complexes For PNGase F treatment, cells were rinsed three times with ice-cold PBS and lysed in 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.5 % NP-40, 0.5 % DOC. Equal amounts of protein (determined using the BCA reagent) were

1222 acetone-precipitated. Pellets were resuspended in 1 % SDS, 100 mM 2-mercaptoethanol (4 mg protein/ml), boiled for two minutes and diluted 1:5 (v/v) in digestion buffer (20 mM Hepes (pH 7.2), 5 mM PMSF, 1.5 % CHAPS). Following digestion with PNGase F (1.0 unit/ 20 mg protein; Glyko, Novato, CA) for 16 hours at 30  C, the reaction was terminated by the addition of SDSsample buffer and aliquots were subjected to immunoblot analysis. For PIPLC treatment, cells were washed three times with ice-cold PBS. Digestion with PIPLC (0.5 unit/ml; Molecular Probes, Eugene, OR) was carried out in PBS at 4  C for 40 minutes. Purification of HMM-PrP complexes Approximately 109 adherent N2a cells were crosslinked in the presence of 1 % formaldehyde as outlined above. All subsequent steps were carried out at 4  C. Extracts were diluted ®vefold into 20 mM Tris (pH 8.0), 50 mM NaCl, loaded onto Q-Sepharose HP and the bound protein was eluted with a linear salt gradient of 50 mM to 600 mM salt in 20 mM Tris (pH 8.0), 0.05 % DOC, 0.05 % NP-40. Eluate fractions containing HMMPrP complexes were pooled and subjected to immunoprecipitation using streptavidin agarose beads that were pre-saturated with a 1:1 (w/w) mixture of biotinylated HuM-D13 and biotinylated HuM-D18 and blocked with 5 % bovine serum albumin (BSA) in 20 mM Tris (pH 8.0). After acidic elution (100 mM glycine, pH 2.5), the complex was concentrated via methanol/chloroform precipitation.63 Separation of HMM-PrP complexes from residual recombinant Fabs and uncrosslinked PrP was carried out on a 6 % preparative SDS-PAGE. HPLC/Mass spectrometry (LC/MS/MS) After extraction from the gel, the peptide digests were separated and analyzed by LC/MS. Nano¯ow HPLC was carried out using an ``Ultimate'' HPLC system (LC Packings, San Francisco, CA) equipped with the FAMOS sample introduction system. Experimental conditions were: injection volume, 1 ml; column, 75 mm I.D. C18 PepMap (LC Packings); solvent A, water with 0.05 % formic acid; solvent B, acetonitrile with 0.04 % formic acid; and gradient, 5-50 % B over 30 minutes at a ¯ow rate of 250 nl/minute. The column ef¯uent was coupled directly via a 25 mm I.D. fused silica capillary transfer line to a QSTAR hybrid tandem mass spectrometer (Applied Biosystems/MDS Sciex, Concord, Ontario, Canada) equipped with a MicroIonSpray source. The progress of each LC/MS run was monitored by recording the total ion current (TIC) as a function of time for ions in the 300 to 1800 m/z range, with a typical spectrum accumulation time of four seconds. Using m/z values identi®ed from an earlier LC/MS run, peptide ions were selected for collision induced dissociation (CID) employing nitrogen collision gas. Peptides eluted over a 15-minute interval with typical chromatographic peak widths of 30 to 45 seconds, which was suf®cient for MS and MS/MS spectra to be acquired and averaged. The isotope spacing was examined for peaks of interest to identify the charge state for each ion. Peak lists were generated manually and submitted to the database searching programs within the web-based package Protein Prospector (http://prospector.ucsf.edu), using MS-Fit for peptide mass mapping and MS-Tag to identify proteins based on the CID spectra.

PrP Interacts with N-CAM Multi-pin peptide library The procedure for the synthesis of multi-pin peptide libraries has been described.64 In short, polypropylene pins were arranged on a solid support block in a 12  8 array. Each synthesis reaction was carried out in a reaction tray with a well for each pin. Individual pins provided multiple reaction sites consisting of a chemical spacer that terminated in a 9-Fmoc protection group. In each cycle in the synthesis, the Fmoc amino-terminal protecting group was removed, the pins were washed, then an activated Fmoc-amino acid was added followed by a second washing step. Upon completion of the synthesis, the amino-termini of the peptides were acetylated and protecting groups were removed with tri¯uoroacetic acid, leaving the unprotected peptide chains attached to the pins. The library consisted of 140 different peptides, each 15 amino acid residues long. Consecutive peptides were shifted along the primary structure of N-CAM by ®ve amino acids and therefore displayed a ten amino acid residue sequence overlap. Non-speci®c binding sites were saturated with blocking buffer (1 % BSA in 0.05 % Tween-20 in PBS, pH 7.2) for two hours at RT. Incubations with full-length mouse rPrP and primary antibody HuM-D13, each at concentrations of 1 mg/ml, were carried out in blocking buffer overnight at 4  C. Between incubations, the library was washed six times, ®ve minutes each, with 0.05 % Tween20 in PBS. The signal development was based on the ¯uorescent peroxidase substrate QuantaBlu (Pierce, Rockford, IL) with an emission maximum at 420 nm. Production and inoculation of N-CAMÿ/ÿ mice N-CAMÿ/ÿ mice were generated by the insertion of the bacterial lacZ gene into the 30 end of the ®rst exon of the N-CAM gene. Consequently, instead of N-CAM, these mice express b-galactosidase driven by the N-CAM promoter.65 RML prions66 were previously passaged in Swiss mice. Mice were inoculated intracerebrally with 30 ml of a 10 % brain homogenate in PBS using a 27gauge needle inserted into the right parietal lobe. Criteria for diagnosis of CNS dysfunction in mice have been described.67

Acknowledgments This work was supported by grants from the National Institutes of Health (NS14069, AG02132, NS39837 and AG10770) as well as by a gift from the G. Harold and Leila Y. Mathers Foundation. LC/MS was carried out in the UCSF Mass Spectrometry Facility, supported by NIH RR01614 and RR12961. We are grateful to David Maltby for technical assistance. Support for G.S.-U. was provided by the Ernst Schering Research Foundation (Berlin, Germany).

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Edited by P. E. Wright (Received 31 July 2001; received in revised form 12 October 2001; accepted 12 October 2001)