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 (NCT_m) 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 (NCT_i) 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. NCT_m also reacted weakly with this lectin. Furthermore, NCT_m 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.

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