Abstract
The cellular prion protein (PrPC) is a glycoprotein that is processed through several proteolytic pathways. Modulators of PrPC proteolysis are of interest because full-length PrPC and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases. PrPC may also act as a receptor for neurotoxic, oligomeric species of other proteins that are linked to neurodegeneration. Importantly, the PrPC C-terminal fragment C1 does not contain the reported binding sites for these oligomers. Western blotting would be a simple end point detection method for cell-based screening of compound libraries for effects on PrPC proteolysis or overall expression level. However, traditional Western blotting methods provide unreliable quantification and have only low throughput. Consequently, we explored capillary-based Western technology as a potential alternative; we believe that this study is the first to report analysis of PrPC using such an approach. We successfully optimized the detection and quantification of the deglycosylated forms of full-length PrPC and its C-terminal cleavage fragments C1 and C2, including simultaneous quantification of β-tubulin levels to control for loading error. We also developed and tested a method for performing all cell culture, lysis, and deglycosylation steps in 96-well microplates prior to capillary Western analysis. These advances represent steps along the way to the development of an automated, high-throughput screening pipeline to identify modulators of PrPC expression levels or proteolysis.
Highlights
The cellular prion protein (PrPC) is a glycoprotein that is processed through several proteolytic pathways
Modulators of PrPC proteolysis are of interest because full-length PrPC and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases
Given that such oligomers are linked to the neurodegeneration observed in Alzheimer’s and Parkinson’s diseases, respectively, and that the putative PrPC binding sites are all absent in C1, compounds that modify PrPC expression or proteolysis may be protective against these disorders
Summary
PrPC, cellular prion protein; GPI, glycophosphatidylinositol; FL, full-length; recPrP, recombinant prion protein; PNGase F, peptide-N-glycosidase F; MW, molecular weight; %CV, percentage coefficient of variation; trxA, thioredoxin A; HRP, horseradish peroxidase; TBS, tris-buffered saline. Capillary Western technology has already been put to use in the neurodegeneration field, including for analyzing the expression of the microtubule-associated protein Tau in brain homogenates [14] and detecting biomarkers to diagnose neurodegenerative diseases [15, 16]. In this manuscript, we outline our progress in optimizing the detection and quantification of the GPI-linked glycoprotein PrPC and its cleavage fragments using capillary Western technology. We report on work to optimize the deglycosylation of PrPC in cell lysates, including the development of an “inplate” format to process samples for capillary Western analysis
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