Identification of prion protein binding proteins by combined use of far-Western immunoblotting, two dimensional gel electrophoresis and mass spectrometry

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Abstract

The cellular prion protein (PrPC), a highly conserved glycoprotein predominantly expressed by neuronal cells, can convert into an abnormal isoform (PrPSc) and provoke a transmissible spongiform encephalopathy. In spite of many studies, the physiological function of PrPC remains unknown. Recent findings suggest that PrPC is a multifunctional protein participating in several cellular processes. Using recombinant human PrP as a probe, we performed far-Western immunoblotting (protein overlay assay) to detect cellular PrPC interactors. Brain extracts of wild-type and PrP knockout mice were screened by far-Western immunoblotting for PrP-specific interactions. Subsequently, putative ligands were isolated by 2-DE and identified by MALDI-TOF MS, enabling identification of heterogeneous nuclear ribonucleoprotein A2/B1 and aldolase C as novel interaction partners of PrPC. These data provide the first evidence of a molecule indicating a mechanism for the predicted involvement of PrPC in nucleic acid metabolisms. In summary, we have shown the successful combination of 2-DE with far-Western immunoblotting and MALDI-TOF MS for identification of new cellular binding partners of a known protein. Especially the application of this technique to investigate other neurodegenerative diseases is promising.

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