Analysis of the Glycosylation Profile of Disease-Associated Water-Soluble Prion Protein Using Lectins

The disease-associated water-soluble form of hamster prion protein (ws-PrP) has recently been found to be less stable than classical PrP. 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-PrP and classical PrP of hamster which might account for the ws-PrP minor stability compared with that of the classical PrP. Thus, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (S) and high-speed pellet (P)] 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-PrP from S and plasma compared with classical PrP from P. These differences have been shown to be potentially responsible for the instability of ws-PrP. Treatment of infected blood with GdnHCl significantly (P<0.01) increased the detection of ws-PrP in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrP stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrP 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-PrP/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrP to convert to ws-PrP both in blood and the brain. Therefore, PrP glycosylation characteristics may provide a tool for the determination risk of prion transmissibility.