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

Figure 2

Analysis of the glycosylation profile of ws-PrP and classical PrP and effect of treatment with GdnHCl. ((a)-(c)) Glycosylation profile of from high-speed supernatant () and plasma is similar: ((a) and (b)) lectin-binding profile to single glycans in captured by 3F4 and 6H4, respectively, from and plasma; (c) glycans total content in captured by 3F4 and 6H4 from and plasma. (d) Comparison between signals of captured by specific anti-PrP polyclonal antibody from 263K-infected hamster plasma and signals of immunoglobulin (IgG) prepared from 263K-infected hamster plasma, before and after treatment with guanidine hydrochloride (GdnHCl) in ELISA. Signals were detected by 6H4 antibody/secondary antibody or only with secondary antibody. (e) Representative silver-stained 12% SDS-PAGE gel of hamster plasma total proteins showing effect of GdnHCl on high-abundance proteins removal from blood. ((f)-(m)) Glycosylation profile of ws-PrP differs from that of classical PrP: ((f) and (g)) lectins binding profile to single glycans in captured by 3F4 and 6H4, respectively, from and high-speed pellet (); (h) glycans total content in captured by 3F4 and 6H4 from and ; (i) percentage of glycans total content in    with respect to that in ; (j) lectins binding profile to glycans in captured by 3F4 from noninfected , plasma and ; ((k) and (l)) lectins binding profile to single glycans in captured by 3F4 from (k) and (l) before and after treatment with GdnHCl; (m) glycans total content in captured by 3F4 from and before and after treatment with GdnHCl. Total proteins from plasma, and were loaded into 96-well ELISA plates that were precoated with the 3F4 or 6H4 antibodies, and the lectin-binding profile was determined by staining with ConA, Concanavalin A; WGA, wheatgerm agglutinin; RCA, Ricinus communis agglutinin; DBA, Dolichos biflorus agglutinin; PNA, peanut agglutinin; SBA, soybean agglutinin; and UEA-1, Ulex europaeus agglutinin I. The sizes of molecular mass markers in kilodaltons are indicated on the left. Data are means ± SD and are representative of at least three independent assays and three different preparations, performed in duplicate. P<0.05, P<0.01, P<0.001, P<0.0001.

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