Identification of prion protein binding proteins by combined use of far-Western immunoblotting, two dimensional gel electrophoresis and mass spectrometry.
The cellular prion protein (PrP(C)), a highly conserved glycoprotein predominantly expressed by neuronal cells, can convert into an abnormal isoform (PrP(Sc)) and provoke a transmissible spongiform encephalopathy. In spite of many studies, the physiological function of PrP(C) remains unknown. Recent findings suggest that PrP(C) 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 PrP(C) 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 PrP(C). These data provide the first evidence of a molecule indicating a mechanism for the predicted involvement of PrP(C) 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.
Strom
Alexander
A
Molecular Medicine, Ottawa Health Research Institute, Lab N1, Box 221, 501 Smyth Road, Ottawa, Ontario K1H 8L6, Canada. astrom@ohri.ca
Diecke
Sebastian
S
Hunsmann
Gerhard
G
Stuke
Andreas W
AW
D000818:Animals; D026481:Blotting, Far-Western / Q000379:methods*; D001921:Brain / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000378:metabolism; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011328:Prions / Q000378:metabolism*; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011328:Prions