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Interactome Analyses Identify Ties of PrP C and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones

Interactome Analyses Identify Ties of PrP C and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones Joel C. Watts 1,2. a, Hairu Huo 1., Yu Bai 1. b, Sepehr
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Interactome Analyses Identify Ties of PrP C and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones Joel C. Watts 1,2. a, Hairu Huo 1., Yu Bai 1. b, Sepehr Ehsani 1,2., Amy Hye Won 1,2, Tujin Shi 1, Nathalie Daude 3, Agnes Lau 3, Rebecca Young 4, Lei Xu 4, George A. Carlson 4, David Williams 5, David Westaway 3, Gerold Schmitt-Ulms 1,2 * 1 Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Ontario, Canada, 2 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada, 3 Alberta Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada, 4 McLaughlin Research Institute, Great Falls, Montana, United States of America, 5 Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada Abstract The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP C ) to diseaseassociated isoforms has remained enigmatic. A quantitative investigation of the PrP C interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrP C paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP C and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP C with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP Sc. A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP C organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins. Citation: Watts JC, Huo H, Bai Y, Ehsani S, Won AH, et al. (2009) Interactome Analyses Identify Ties of PrP C and Its Mammalian Paralogs to Oligomannosidic N- Glycans and Endoplasmic Reticulum-Derived Chaperones. PLoS Pathog 5(10): e doi: /journal.ppat Editor: Neil Mabbott, University of Edinburgh, United Kingdom Received March 16, 2009; Accepted September 8, 2009; Published October 2, 2009 Copyright: ß 2009 Watts et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work on this project was funded through grant support from the Canadian Institutes of Health Research (CIHR; MOP (GSU) and MOP (DW)). GSU received support from the W. Garfield Weston Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * a Current address: Institute for Neurodegenerative Diseases, University of California, San Francisco, California, United States of America b Current address: Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China. These authors contributed equally to this work. Introduction Prions are the causative agents underlying a range of rare but invariably fatal neurodegenerative diseases in humans and other mammals. In disease, the cellular prion protein (Prnp; herein referred to as PrP C ) converts to a disease-associated conformer (PrP Sc ) with different physicochemical properties [1]. PrP C is a relatively small protein which assembles into an unstructured N- terminal domain and a globular C-terminal half characterized by the presence of an internal disulfide bridge, up to two N-linked glycans and a glycosylphosphatidylinositol (GPI) anchor for insertion into the cellular plasma membrane. Multiple lines of investigation have led to the conclusion that mature PrP C is embedded in specialized membrane domains, so-called raft-like domains, rich in cholesterol and sphingolipids [2]. It has been suggested that these raft-like domains host the self-perpetuated accumulation of PrP Sc which subsequently triggers a poorlyunderstood cascade of events that ultimately leads to cell death. Whereas significant progress has been made in the past few years in defining the minimal requirements for PrP C conversion in vitro [3], the molecular environment which hosts the earliest steps in prion disease manifestation in neuronal cells remains enigmatic. This shortcoming is not due to a lack of proteins proposed to interact directly with PrP C. In fact, more than three dozen proteins have been suggested to reside in spatial proximity to PrP C using multiple experimental paradigms [4]. Additional candidate interactors have been proposed to bind preferentially to PrP Sc [5,6,7]. In surveying this body of literature, however, it is apparent that very few of the candidate interactors have been independently verified by multiple investigators and, overall, little agreement exists as to their relative importance for prion protein biology. Notable exceptions may represent the 37-kDa/67-kDa laminin receptor precursor (herein referred to as LRP; also known as ribosomal protein SA (Rpsa), and not to be confused with Lrp1, the low density lipoprotein receptor-related protein 1) [8], one of the first proteins identified to bind to the prion protein in a yeast PLoS Pathogens 1 October 2009 Volume 5 Issue 10 e Author Summary Prions underlie rare but invariably fatal neurodegenerative diseases in humans and other mammals. Public awareness of these diseases has grown with the occurrence of cases of BSE (also known as Mad Cow Disease ) and the realization that this disease can, in rare instances, be transmitted to humans. The normal cellular prion protein is found in most cell types within the body. In disease, this protein acquires a different shape which tends to aggregate and poison nearby cells. The disease-associated conversion of the prion protein appears to require its localization to specialized cellular membrane regions rich in cholesterol; however, the precise molecular environment which hosts this event has remained elusive. We used a cell-based disease model to identify proteins which reside in close proximity to the prion protein and two closely related mammalian proteins. We demonstrate that these three proteins may not only populate highly similar environments but may also interact with each other. We further identified an extended molecular network in proximity of these proteins which supports functions in adhesion control, lactate metabolism and cell fusion events. It is anticipated that these insights will contribute to efforts to rationally interfere with aberrant proteinprotein interactions underlying these diseases. two-hybrid (Y2H) screen [9]; the neural cell adhesion molecule (Ncam1; herein referred to as NCAM), which was initially identified in a cellular crosslinking study based on a coimmunoprecipitation methodology [10]; and heparin sulfate proteoglycans (HSPGs) [11,12]. Since its original discovery, LRP has been proposed to act as a cell surface receptor for PrP C that may play a role in prion propagation [13]. In the case of NCAM, binding to PrP has been shown to play a role in neuritic outgrowth possibly mediated through an interaction with Fyn tyrosine kinase [14,15]. In studying the molecular environment of membrane proteins, a limitation exists in that the proteins must be solubilized by the addition of detergents in a manner that does not disrupt the protein-protein interactions under investigation. A solution to this obstacle constitutes the covalent stabilization of interactions by chemical crosslinking prior to the disruption of cellular integrity. In this regard, we previously reported on a large-scale investigation of the molecular PrP C neighborhood in mice following limited in vivo crosslinking by time-controlled transcardiac perfusion (tctpc)- based delivery of formaldehyde (FA) to the brain [16]. A conspicuous feature of this PrP C interactome dataset was the relative abundance of membrane proteins which harbor immunoglobulin (Ig)-like folding motifs in their extracellular domains confirming previous data describing a binding domain of PrP C within these folds [10]. Given the large diversity of cell types present in the brain, we questioned whether an equivalent concentration of Ig-like domainharboring proteins would have been found if the investigation of the PrP C interactome had been restricted to a single cell type. A particular concern was that the relative abundance of Ig-like domain-harboring proteins in the previous dataset may have masked the ability to identify biologically important interactions of PrP C that are more transient in nature or involve less abundant proteins. We therefore chose to reinvestigate the PrP C interactome in mouse neuroblastoma cells (N2a), which are by far the bestcharacterized cell model for the study of the biology and conversion of PrP C to PrP Sc [17,18]. To facilitate the discrimination of unspecific binders from specific interactors in this study, we incorporated quantitative mass spectrometry based on isotopic labeling and extended our investigation to the mammalian PrP C paralogs Doppel (encoded by the Prnd gene; protein product herein referred to as Dpl) and Shadoo (encoded by the Sprn gene; protein product herein referred to as Sho). To avoid the use of non-identical affinity chromatography steps, we equipped the three bait proteins with the same N-terminal FLAG epitope [19], with the awareness that a similar PrP C expression construct had in prior investigations neither interfered with the posttranslational processing nor the conversion of PrP C in cell or animal models [18]. We now present data which suggest that members of the mammalian prion protein family may populate highly similar molecular environments when expressed in the neuroblastoma cell model system. We further document that a subset of endoplasmic reticulum (ER) chaperones which interact strongly with PrP C escape from the ER to reside in spatial proximity to PrP C at the plasma membrane. Pharmacological inhibition of these chaperones could increase PrP Sc levels in prion-infected N2a sublines, suggesting a protective role for the chaperones in this paradigm. Finally, our data consolidate multiple previously controversial interactions and suggest a scenario whereby PrP C may organize its molecular environment by its ability to recognize a specialized subset of cell adhesion molecules which recruit membrane proteins carrying high-mannose glycans into spatial proximity with PrP C. Results Large-scale quantitative and comparative interactome investigation of members of the mammalian prion protein family In preparation for this study, mouse neuroblastoma cells (N2a) stably expressing N-terminally FLAG-tagged full-length versions of Dpl, PrP and Sho were generated (Fig. 1). Small-scale expression tests followed by diagnostic N-glycosidase F or phosphatidylinositol-specific phospholipase C digestions confirmed that all three bait proteins mimicked their untagged parent molecules in (i) the presence of N-glycans, and (ii) membrane attachment by means of a GPI anchor [20] (and data not shown). To generate the biological source material for a large-scale comparative interactome investigation, the three cell lines and an empty-vector stably-transfected N2a cell line serving as a negative control were expanded to 10 9 cells each using cell culture conditions which promote adherent growth. To covalently stabilize protein-protein interactions prior to the disruption of cellular integrity, cells were subjected to a 15-min treatment with FA [10]. Subsequently, cells were lysed by the addition of detergents and the four extracts (each containing approximately 500 mg of cellular protein) purified sideby-side on anti-flag affinity agarose matrices. The presence of covalent linkages between proteins permitted the use of highly stringent washing conditions to minimize the presence of unspecific binders. Following elution from the affinity matrix (with a yield of approximately 100 mg of protein material per sample), protein complexes were denatured and trypsinized. Finally, peptide mixtures were tagged with isobaric tags for relative and absolute quantitation (itraq) [21], and the samples were combined and subjected to a comprehensive analysis by tandem mass spectrometry (MS/MS) (Fig. 2). A query of mouse protein databases led to the identification of more than 100 proteins. All identifications can be considered confident by multiple measures: (i) identical identifications were made by two matching algorithms; (ii) scores assigned by the algorithms exceeded significance thresholds (P,0.05) for all identifications and were based on a minimum of two peptides; and (iii) a search of a decoy database generated by the inversion of sequences for all PLoS Pathogens 2 October 2009 Volume 5 Issue 10 e Figure 1. Expression analysis of FLAG-tagged mouse prion proteins. A, Schematic representation of murine prion proteins with FLAG tags inserted near the N-terminus. B, Expression of transiently-transfected FLAG-prion proteins in N2a cells as assessed by Western blotting with the anti- FLAG M2 antibody. The presence of a non-specific band in N2a lysates recognized by the M2 antibody is denoted by an asterisk. doi: /journal.ppat g001 mouse protein entries resulted in no identifications which shared any of the above features [22]. A reduction of the total list of identified proteins to the subset of proteins whose identification correlated with the presence of at least one of the three bait proteins was based on peak intensities of itraq reporter ions found in the low mass range of individual collision induced Figure 2. Flow chart depicting strategy for semi-quantitative comparison of prion protein family interactomes. In vivo formaldehyde crosslinked protein complexes containing N-terminally FLAG-tagged bait proteins are stringently purified on anti-flag agarose parallel to a negative control sample derived from an empty vector expression clone. Following alkylation, reduction and trypsinization, digests are side-by-side itraq labeled and subsequently combined. Two-dimensional liquid chromatography of peptides is coupled to online ESI-MS/MS, which is followed by computationally-aided protein identification and quantitative analysis. doi: /journal.ppat g002 dissociation (CID) spectra. The relative intensity of these ions is indicative of the relative contribution of each of the itraqlabeled samples to the generation of a given CID spectrum. Thus, by calculating the ratio of reporter ion intensities for each of the three bait-specific reporter ions (itraq 115: Dpl; itraq 116: PrP; itraq 117: Sho) and the negative control ion (itraq 114 reporter), peptides purifying with at least one of the three baits were recognized by itraq reporter ion ratios greater than 1. The above analysis revealed that more than 50 proteins co-purified with at least one of the three bait proteins (Table 1). Interestingly, the majority of these proteins co-purified with all three bait proteins, suggesting that the molecular environment of the three members of the mammalian prion protein family assessed in the context of mouse neuroblastoma cells is highly similar. We next explored the known cellular localization of candidate interactors based on bioinformatic methods and literature mining. This investigation revealed that multiple proteins in the dataset were likely to encounter the three bait proteins during their early passage through the secretory pathway, since they constitute (i) classical ER chaperones (heat shock protein 5, Hspa5; calnexin, Canx; calreticulin, Calr; endoplasmin, Hsp90b1), (ii) isomerases which facilitate disulfide or proline cis-trans rearrangements (protein disulfide isomerase associated 3, Pdia3; protein disulfide isomerase associated 4, Pdia4; prolyl 4-hydroxylase beta polypeptide, P4hb; peptidylprolyl isomerase B, Ppib), or (iii) proteins involved in the trafficking between ER and Golgi compartments (transmembrane emp24 transport domain containing 9, Tmed9; and transmembrane emp24-like trafficking protein 10, Tmed10). Other proteins in the dataset were likely to reside in spatial proximity to the mature bait proteins at the plasma membrane, because they are themselves known to be either (iv) embedded in the plasma membrane through transmembrane (TM) domains (NCAM; transferrin receptor, Tfrc; integrins; neuropilin 1, Nrp1; L1 cell adhesion molecule, L1cam; basigin, Bsg), or constitute (v) secreted proteins (galectin-1, Lgals1; family with sequence similarity 3, member C, Fam3c; ectonucleotide pyrophospha- PLoS Pathogens 3 October 2009 Volume 5 Issue 10 e Table 1. Prion protein family interactome in mouse neuroblastoma cells. IPI accession number Symbol Identified proteins a Pept. b Unique c % Cov. d Control e Dpl PrP Sho IPI:IPI Prnd prion protein dublet (doppel, Dpl) IPI:IPI Prnp prion protein (PrP) IPI:IPI Sprn shadow of prion protein (shadoo, Sho) IPI:IPI Ldha lactate dehydrogenase A IPI:IPI Pdia3 protein disulfide isomerase associated IPI:IPI Lgals1 galectin-1 (lectin, galactose binding, soluble 1) IPI:IPI Ncam1 neural cell adhesion molecule 1 (NCAM) IPI:IPI Gm9234 predicted gene 9234 (EG668548) IPI:IPI Gap43 growth associated protein 43 (neuromodulin) IPI:IPI Rpsa ribosomal protein SA (laminin receptor precursor, LRP) IPI:IPI Hspa5 heat shock protein IPI:IPI H2-K1 histocompatibility 2, K1, K region IPI:IPI Ftl1 ferritin light chain IPI:IPI Fam3c family with sequence similarity 3, member C IPI:IPI P4hb protein disulfide-isomerase (prolyl 4-hydroxylase beta) IPI:IPI Ppib peptidylprolyl isomerase B IPI:IPI Rps21 ribosomal protein S IPI:IPI H2-D1 histocompatibility 2, D region locus IPI:IPI Canx calnexin precursor IPI:IPI Hsp90ab1 heat shock protein 90 alpha (cytosolic), class B member IPI:IPI Calr calreticulin IPI:IPI Ywhaz protein zeta IPI:IPI Bsg basigin IPI:IPI Ywhab protein beta IPI:IPI Tmed2 transmembrane emp24 domain trafficking protein IPI:IPI Pdia6 protein disulfide isomerase associated IPI:IPI Tmed10 transmembrane emp24-like trafficking protein IPI:IPI Ywhag protein gamma IPI:IPI C1qbp complement component 1, q subcomponent binding protein IPI:IPI Tfrc transferrin receptor protein IPI:IPI Hsp90b1 heat shock protein 90, beta (Grp94), member 1 (endoplasmin) IPI:IPI Hyou1 hypoxia up-regulated IPI:IPI Fyn fyn proto-oncogene IPI:IPI Basp1 brain abundant, membrane attached signal protein IPI:IPI Tmem206 transmembrane protein 206 (C1orf75) IPI:IPI Gdi2 guanosine diphosphate (GDP) dissociation inhibitor IPI:IPI Itgb1 integrin beta IPI:IPI Enpp1 ectonucleotide pyrophosphatase/phosphodiesterase IPI:IPI Slc3a2 solute carrier family 3, member 2 (CD98) IPI:IPI Itga6 integrin alpha IPI:IPI Ugcgl1 UDP-glucose ceramide glucosyltransferase-like IPI:IPI Gnao1 guanine nucleotide binding protein, alpha O IPI:IPI Itgav integrin alpha V IPI:IPI Atp1a1 sodium/potassium transporting ATPase alpha 1 polypeptide IPI:IPI Atp1b3 sodium/potassium transporting ATPase beta 3 polypeptide IPI:IPI Gnai2 guanine nucleotide binding protein, alpha inhibiting IPI:IPI Pdia4 protein disulfide isomerase associated IPI:IPI Nrp1 neuropilin IPI:IPI Igsf8 immunoglobulin superfamily, member PLoS Pathogens 4 October 2009 Volume 5 Issue 10 e Table 1. Cont. IPI accession number Symbol Identified proteins a Pept. b Unique c % Cov. d Control e Dpl PrP Sho IPI:IPI Gn
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