Abstract
The disease-associated water-soluble form of hamster prion protein (ws-PrPSc) has recently been found to be less stable than classical PrPSc. Since the stability of PrP to degradation correlates with its glycosylation level, the aim of this study was to investigate whether there are differences between the glycosylation of ws-PrPSc and classical PrPSc of hamster which might account for the ws-PrPSc minor stability compared with that of the classical PrPSc. Thus, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (SHS) and high-speed pellet (PHS)] and blood plasma by anti-PrP antibodies (3F4 and 6H4) and subjected to screening for glycans by lectins under denaturing or nondenaturing procedures in a sandwich lectin-ELISA. Glycans have been found in minor quantities and differently exposed on ws-PrPSc from SHS and plasma compared with classical PrPSc from PHS. These differences have been shown to be potentially responsible for the instability of ws-PrPSc. Treatment of infected blood with GdnHCl significantly (P<0.01) increased the detection of ws-PrPSc in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrPSc stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrPSc glycosylation with particular regard to the mannose sugar. Analysis of lectins immunoreactivity toward total proteins from plasma collected before and at different time points after infection revealed that mannose might exert a stabilizing effect toward all of hamster blood glycoproteins, regardless of scrapie infection. Since low levels of ws-PrPSc/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrPC to convert to ws-PrPSc both in blood and the brain. Therefore, PrPC glycosylation characteristics may provide a tool for the determination risk of prion transmissibility.
Highlights
IntroductionA recent study revealed the existence of a water-soluble form of the prion protein (ws-PrP) in blood plasma and brain of Syrian hamster [3]
Transmissible spongiform encephalopathies (TSEs) or prion diseases are invariably fatal neurodegenerative diseases characterized by the conversion of the cellular prion protein (PrPC: classical PrPC) to the partially protease-resistant form (PrPSc: classical PrPSc, which is the hallmark of prion diseases) and its deposition in the central nervous system [1, 2]
To investigate the glycosylation profile of ws-PrPSc, brain-derived and plasma-derived ws-PrPSc was captured from terminally scrapie-infected hamster highspeed supernatant (SHS) and plasma, respectively, by 3F4 or 6H4 anti-PrP monoclonal antibodies and subjected to screening for glycans by a panel of seven biotinylated lectins in a sandwich lectin-ELISA
Summary
A recent study revealed the existence of a water-soluble form of the prion protein (ws-PrP) in blood plasma and brain of Syrian hamster [3] This PrP has biochemicalphysical properties that are substantially different from those of the classical PrP. [3] displayed a glycotyping that was different from that of the classical PrPC and PrPSc, showing a slightly faster migration mobility and a diglycoslated band with higher propensity to degradation by endogenous enzymes. This increased susceptibility to degradation of ws-PrP compared to the classical PrP may be due to an instability issue caused by glycosylation differences between the two proteins. The oligosaccharide moiety is responsible for many glycoproteins’ functions, such as synthesis, folding, trafficking, stability, recognition, and regulation of the proteins themselves and many of their diverse interactions [5, 6]
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