Interactome analyses identify ties of PrP and its mammalian paralogs to oligomannosidic N-glycans and endoplasmic reticulum-derived chaperones

Abstract

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP(C)) to disease-associated 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.

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.

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.

Figure 3. The mammalian prion protein family interactome.

Network depicting proteins identified in spatial proximity to Dpl, PrP^C and Sho as nodes and their direct or indirect interactions as spokes. Components identified in previous studies by other investigators are shown with red lines surrounding and red edges connecting the nodes. The intensity of gray shading of nodes correlates directly with the fractional sequence coverage with which individual components were identified. Increased spoke thickness indicates stronger co-enrichment with the connected bait protein. Node shapes indicate predominant cellular localization of proteins, i.e. ER and Golgi compartments (rectangular), plasma membrane (oval), cytosol (diamond) or secreted (octagonal). Nodes depicting proteins whose presence in the samples correlated strongest with the presence of a given bait protein are arranged in spatial proximity to the respective bait protein nodes in the bottom half of the network.

Figure 4. Evidence for PrP^C in high molecular weight protein complexes captured by a lectin with specificity for oligomannosidic glycans.

Brain extracts from mice subjected to transcardiac perfusion crosslinking were passed over snowdrop lectin agarose. Western blot analysis of eluate fractions with 7A12 antibody documented presence of PrP^C in high molecular weight complexes (lane 5). Only weak bands were detected when the affinity capture step was preceded by incubation of extracts with soluble snowdrop lectin (sGNA, lane 2). Crosslink reversal by 5-min (lanes 3 and 6) or 20-min (lanes 4 and 7) heat treatment of samples caused a shift of PrP^C reactive bands to levels of fully glycosylated PrP^C consistent with the release of PrP^C from crosslinked complexes.

Figure 5. Evidence for cell surface localization of a subset of PDIs and calreticulin in mouse neuroblastoma cells.

N2a and ScN2a cells were subjected to cell surface biotinylation or mock treatment. Subsequently, cellular extracts were side-by-side affinity purified on a streptavidin agarose matrix. Extracts and streptavidin agarose eluate fractions were analyzed by Western blotting and membranes probed with antibodies directed against histone 2B, PrP, P4hb, Pdia3 and calreticulin. The relative strength of P4hb-, Pdia3- and calreticulin-specific signals present in eluate fractions from biotinylated (lanes 6 and 8) versus non-biotinylated samples (lanes 5 and 7) is consistent with the conclusion that a subset of these proteins resided at the cell surface during the biotinylation step. The relative intensity of P4hb- and Pdia3-derived signals in extract (40 µg total protein loaded per lane) and eluate fractions can be estimated from the concomitant analysis of 1/4 and 1/20 of the extract fraction shown in lane 1. Thus, P4hb and Pdia3 signals in the eluate fraction (captured from 2 mg total extract) were equivalent to or exceeded the amount of these proteins present in 2 µg of extract. Please note the presence of histone 2B in all eluate fractions (lanes 5–8), in support of the conclusion that the protein binds unspecifically to the affinity matrix.

Figure 6. Inhibition of protein disulfide isomerases causes accumulation of PrP^Sc in a subset of ScN2a cell clones.

A, Bacitracin or DTNB, reagents known to inhibit PDIs through orthogonal modes of binding, were added at the indicated concentrations to the cell culture medium and left on the ScN2a cells for a duration of 2 days. Following cell lysis, protein levels were adjusted and a subset of samples subjected to digestion with proteinase K (lanes 411). All samples were analyzed by Western blotting with a PrP specific antibody. Please note the increase in intensity of low molecular weight bands characteristic for the presence of PrP^Sc even without proteinase K treatment (lanes 1–3). B, A two-sample, two-tailed Student's t-test assuming unequal variance was utilized to determine the statistical significance of bacitracin-treated band intensities compared to normalized untreated samples. The result of this analysis indicated a significant increase of PrP^Sc in ScN2a clones 1 and 2 treated with 1 mM bacitracin (P-value = 0.01).