CADASIL mutations impair Notch3 glycosylation by Fringe

Mutations in the NOTCH3 gene trigger adult-onset stroke and vascular dementia in patients with CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). All CADASIL mutations described to date affect the epidermal growth factor-like (EGF-like) repeats located in the extracellular domain of the Notch3 receptor. These domains are also the target of sequential complex O-linked glycosylation mediated by protein O-fucosyltransferase 1 and Fringe. We investigated whether O-fucosylation or Fringe-mediated elongation of O-fucose on Notch3 is impaired by CADASIL mutations. Biochemical studies of a Notch3 fragment containing the first five EGF-like repeats of Notch3, including the mutational hot spot, showed that CADASIL mutations do not affect the addition of O-fucose but do impair carbohydrate chain elongation by Fringe. CADASIL changes also induced aberrant homodimerization of mutant Notch3 fragments and heterodimerization of mutant Notch3 with Lunatic Fringe itself. Together, these data suggest that Fringe plays a role in CADASIL pathophysiology.