Fig. 1. Glycosylation modifications of nicastrin and effect of PS deficiencies. (A)
Western blot analysis of total cell extracts from wild-type,
PS1–/–, PS2–/– and
PS1–/–PS2–/– MEFs using an
antibody raised to the C-terminus of nicastrin (B59.2). Cell extracts were
subject to digestion with endoH (H) and N-glycosidase F (F); –20C and
37C represent control samples (no addition of enzyme) kept at –20°C
and 37°C. Note the partial endoH resistance of mature nicastrin
(NCTm) in wild-type and in PS2–/– MEFs, and
in PS1–/– MEFs to a lesser extent (overexposed part of
the blot below). In PS1–/–PS2–/–
MEFs, only endoglycosidase-sensitive immature nicastrin (NCTi) is
observed (see also overexposed inset). (B) Lectin binding to nicastrin.
Nicastrin was immunoprecipitated from total cell extracts of wild-type MEFs
and blotted using the indicated lectins. The immature glycosylated nicastrin
reacted strongly with Galanthus nivalis agglutinin (GNA), indicating
the presence of terminally linked mannose residues. NCTm also
reacted weakly with this lectin. Furthermore, NCTm reacted with
Maackia amurensis agglutinin (MAA) and Datura stramonium
agglutinin (DSA), indicating the presence of sialic acid residues and complex
and hybrid N-glycan structures, respectively. No reaction was observed with
peanut agglutinin, specific for O-glycan modifications. Control glycoproteins
demonstrated the specificity of the lectins. (C) Western blot analysis of
total cell extracts from wild-type and PS1–/– neurons.
C, control (no enzyme); H, endoH digestion; F, N-glycosidase F digestion; N,
neuraminidase digestion; O, O-glycosidase digestion. Notice the partial
sensitivity to endoH and the complete sensitivity to N-glycosidase F of
neuronal nicastrin. A small shift in mobility is also observed after
neuraminidase digestion. (D) Western blot analysis of total cell extracts from
wild-type and APP–/– MEFs. C, control (no enzyme); H,
endoH digestion; F, N-glycosidase F digestion. No differences between
wild-type and APP–/– MEFs could be observed.