FIGURE 4.

Missense mutations in phosphotransferase Notch repeat 1 impair recognition of α-iduronidase. A, alignment of the Notch repeats 1 and 2 in human αβ phosphotransferase (PT) and the Lin12-Notch repeats (LNR) of human Notch 1. Indicated in bold are the cysteines that are mutated in MLIII αβ patients, and the different colors show disulfide bond formation. B, confocal immunofluorescence microscopy shows normal Golgi localization for the Notch 1 mutants (C442Y, C461G, and C468S) but partial Golgi/ER localization for the Notch 2 mutants (C505Y and C523R). Scale bars, 10 μm. C, HEK293 cell lysates expressing WT or mutant αβ phosphotransferase were subjected to SDS-PAGE and anti-V5 immunoblotting ∼48 h after transfection. Although the Notch 1 mutant β subunit levels were comparable with WT, the Notch 2 mutants showed reduced levels of β subunit, which is consistent with impaired ER exit. The anti-GAPDH control shows that the expression levels of the mutants were similar. D and E, although the activity toward α-MM (D) is normal with the Notch 1 mutants, the activity toward α-iduronidase (IDU, E) was significantly decreased (*, p < 0.05, Student's t test). The activity of the Notch 2 mutants C505Y and C523R was reduced for both α-MM (**, p < 0.01) and α-iduronidase. The values show the activity as a percentage of WT enzyme activity (averages ± S.E.) of at least three independent experiments, measured in duplicate.