Table 1.
Subcellular localization and substrate specificity of mammalian sialidases.
Table 2.
Primers for qRT-PCR (F—forward, R—reverse).
Fig 1.
Distribution of glycoforms of full-length PrPC, C1 and C2 fragments on 2D blots.
(a) Schematic diagram showing full-length PrPC and the sites of alternative cleavage that generate C-terminal fragments C1 or C2. Positions of epitopes for 3F4 and SAF-84 antibody relative to the cleavage sites are indicated. Sialic acid residues on the N-linked glycans and the GPI-anchor are shown by a diamond symbol. (b) 2D blot analysis of brain homogenate from Syrian hamster stained with 3F4 or SAF-84 antibody, or brain homogenate from wild type mice stained with SAF-84 antibody. Syrian hamster brain material was used to map location of full-length PrPC, C1 and C2 fragments on 2D because antibodies toward N-terminal region of mouse PrP gave poor resolution.
Fig 2.
2D analysis of PrPC and C1 in Neu1-/- mice.
2D Western blotting of brain homogenates from FVB Neu1-/- (a) and corresponding FVB wild type control mice (b), or C57BL6 Neu1-/- (c) and corresponding C57BL6 wild type control mice (d). 2D blots of three individual brains are shown in panel (a). 2D analysis of protein markers is provided as a reference in panel e. Filled black arrowheads mark diglycosylated full-length PrPC (FL), whereas three arrows mark di-, mono- and unglycosylated forms of C1. Blots were stained with SAF-84 antibody.
Fig 3.
2D analysis of PrPC and C1 in Neu3-/-, Neu4-/- and Neu3-/-/Neu4-/- mice.
2D Western blotting of brain homogenates from Neu3-/- (a), Neu3-/-/Neu4-/- (b), Neu4-/- (c) and C57Bl6 wild type control mice (d). 2D blots of three individual brains are shown for each group. Filled black arrowheads mark diglycosylated full-length PrPC (FL), whereas three arrows mark di-, mono- and unglycosylated forms of C1. Blots were stained with SAF-84 antibody.
Fig 4.
Relationship between C1 fragments and full-length PrPC.
SDS-PAGE followed by Western blotting of brain materials from Neu1-/- (a), Neu3-/- (b), Neu4-/- (c), Neu3-/-/Neu4-/- (d) and corresponding wild type controls for each group of knockout mice. Replicas within each animal groups correspond to separate mouse brains. Filled black arrowheads mark diglycosylated full-length PrPC (FL), whereas arrows mark diglycosylated C1. (e, f) Ratio of diglycosylated C1/ dyglycosylated full-length PrPC from densitometry analysis for FVB Neu1-/- and corresponding FVB wild type mice (e), or C57BL6 Neu3-/-, Neu4-/-, Neu3-/-/Neu4-/- and corresponding C57BL6 wild type mice (f) is plotted for each animal group. (g) The total amounts of both full-length PrPC and C1 fragments in Neu1-/-, Neu3-/-, Neu4-/- and Neu3-/-/Neu4-/- and corresponding wild type controls. The signal intensity in each group is normalized relative to the intensity of corresponding wild type controls. The data represent means ± SD from three mouse brains for each group.* indicate significant differences (P<0.05, n = 3), whereas # indicate lack of significant differences (P>0.05, n = 3).
Fig 5.
Inhibition of sialidases does not alter sialylation patterns in N2a cells.
(a) The mRNA levels for Neu1, Neu3, and Neu4 in N2a cells were normalized to the glyceraldehyde 3-phosphate dehydrogenase internal control. Mean +/- Standard Deviation (n = 4) are shown. (b) Western blotting of N2a cell lysate (lane 2) demonstrating large amounts of the C1 proteolytic fragment. Molecular weight markers are shown on lane 1. (c) 2D Western blotting of cell lysate of N2a cells cultured in the absence of neuraminidase inhibitor (lower panel) or in the presence of 5 mM DANA for 2 hours (upper panel). Filled black arrowheads mark diglycosylated full-length PrPC (FL), whereas three arrows mark di-, mono- and unglycosylated forms of C1. Blots were stained with SAF-84 antibody. Three independent experiments were performed.
Fig 6.
Inhibition of sialyltransferases alters sialylation pattern in N2a cells.
(a) The mRNA levels for ST3Gal3, ST3Gal4, ST3Gal6, ST6Gal1 and ST6Gal2 in N2a cells were normalized to the glyceraldehyde 3-phosphate dehydrogenase internal control. Mean +/- Standard Deviation (n = 4) are shown. (b,c) 2D Western blotting of cell lysate of N2a cells cultured in the absence of sialyltransferase inhibitor (b) or in the presence of 256 μM 3Fax-Neu5Ac for 24 hours (c). Filled black arrowheads mark diglycosylated full-length PrPC (FL), whereas three arrows mark di-, mono- and unglycosylated forms of C1. Blots were stained with SAF-84 antibody. The experiment was performed twice with two independent repeats each time. 2D blots from each independent experiment are shown. (d) Sialylation profiles of diglycosylated C1 fragment isoforms for N2a cells cultured with 3Fax-Neu5Ac (Fax, blue) and corresponding control cells (pink), or N2a cells cultured with DANA (red) and corresponding control cells (green). Sialylation profile of N2a cells treated with A.ureafaciens sialidase (Neu, black) is provided as a reference.
Fig 7.
Hypothetical mechanism of PrPC degradation.
Two alternative degradation pathways are proposed. The first pathway involves α-cleavage as a first step, which produces C1 fragment. Following α-cleavage, the C1 fragment undergoes desialylation by NEU1, a process that triggers its very fast degradation. The model predicts that desialylation of C1 is required for its degradation, therefore NEU1 deficiency leads to accumulation of C1. According to the second pathway, full-length PrPC is desialylated directly in the absence of α-cleavage. The C-terminal folded domain and C1 are depicted as circles; sialic acid residues are shown as diamonds.