Correction: Interactome Analyses Identify Ties of PrPC 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 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.

Membrane Domain Localization and Interaction of the Prion-Family Proteins, Prion and Shadoo with Calnexin

“…The two bands seen here by us could correspond to these two glycosylated forms of Sho, when tagged by EYFP in our N2a cells. The levels of endogenous Sho in N2a cells are known to be below detection limits by Western blot of the cell lysates [21,65] and as also observed by us (data not shown). Contrarily, the endogenous PrP C is well detected in parental N2a (Supplemental Figure S5D) and in transgenic PrP-EGFP (data not shown) cells and appears as multiple bands based on its different glycosylation states ranging between 26 and 42 kDa (Supplemental Figure S5D, lanes 1 and 2), as commonly observed [75,83].…”

“…We chose mouse neuroblastoma Neuro-2a (N2a) cells as a model system, since they had widely been used in the past for the study of the biology and conversion of PrP C [79], as well as for mapping the interactome of prion paralogs [21] and, therefore, are bestcharacterized for prion studies. To monitor the two prion family proteins, Shadoo (Sho) and prion protein (PrP), we developed N2a cells stably expressing either of these proteins in fusion with a fluorescent protein tag (EYFP for Sho and EGFP for PrP) at their Ctermini, preceding their GPI-anchor signal peptides (termed Sho-EYFP and PrP-EGFP cells, respectively).…”

“…Its presence, however, is required for the acquisition of prion disease, as Prnp-KO mice are resistant to propagating infection and developing disease after intracerebral inoculation by infectious prions [20]. Furthermore, a multitude of binding partners had been reported for PrP C , including protein and nonprotein interactors [21,22], rendering it a multifaceted and multitasking protein involved in several cellular processes: stress protection, metal ion homeostasis, cell differentiation, adhesion, neuronal growth, myelin maintenance, mitochondrial homeostasis, circadian rhythm and immune modulation [23,24]. Importantly, contrary to PrP Sc , PrP C is mostly invoked in neuroprotective roles, exerted either directly or indirectly, participating via binding partners in cell signaling processes.…”

“…However, its absence in Sprn 0/0 or in double-knockout (Sprn 0/0 -Prnp 0/0 ) mice proved otherwise, resulting in no dramatic phenotypes [52,53] and rendering Sho's cellular role also puzzling. Many candidate interacting partners had been proposed for Sho as well [21], among them several common with PrP; moreover, they are confirmed binding partners themselves [54,55]. All of these hint at a less univocal cellular role for Sho, perhaps similarly to PrP; however, further studies and/or confirmation of more of its proposed binding partners and in different systems is needed in order to clarify this.…”

Crystallization and preliminary X-ray diffraction analysis of a specific VHH domain against mouse prion protein

“…Notable examples of TSEs include bovine spongiform encephalopathy (BSE), commonly known as mad cow disease; in humans, it is known as new variant Creutzfeldt-Jakob disease (CJD). These disorders are associated with the conversion of the normal cellular prion protein PrP C to its pathogenic isoform PrP Sc (Watts et al, 2009). Prion diseases are characterized by long incubation periods, spongiform changes and the accumulation of misfolded protein deposits.…”