Mapping O-Fucosylation and O-Glucosylation on Human Notch3 Epidermal Growth Factor-Like (EGF) Repeats Using Nano-LC-ESI-MS/MS
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The Notch protein is a transmembrane signaling protein responsible for regulating several important pathways among all metazoans including cell proliferation, differentiation, and death. Notch exists as one protein in Drosophila, but has four homologs in mice and humans (Notch1- 4). A defining component of the Notch protein is the presence of 29-36 tandem epidermal growth factor-like (EGF) repeats, small protein motifs defined by the presence of six cysteines forming three disulfide bonds. Defects in Notch have been linked to various diseases like Alagille syndrome and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). CADASIL is responsible for early onset of dementia in patients aged 40-50, along with migraines and stroke. CADASIL is characterized by the presence and accumulation of granular osmiophilic material (GOMs) and Notch3 extracellular domain in close proximity to vascular smooth muscle cells, eventually leading to the degradation of vascular smooth muscle cells. Previous evidence showed that mutations in Notch3 are related to the pathogenesis of CADASIL, mainly in EGF repeats 1-5 and 10-11. A majority of these mutations have been identified as missense mutations leading to the addition or deletion of cysteine residues, which is vital component of O-linked glycosylation in Notch. The two main types of O-linked glycosylation on Notch are O-fucosylation initiated at the consensus sequence C2XXXX(S/T)C3 by Pofut1, and O-glucosylation initiated at the consensus sequence C1XSX(P/A)C2 by Poglut1/Rumi, where C1-3 are three of the 6 conserved cysteines within the EGF repeat. After O-fucosylation, the O-fucose can be elongated by Fringe, which adds a GlcNAc to the fucose, followed by subsequent additions by other enzymes, resulting in the tetrasaccharide Siaα 2-3Galβ 1-4GlcNAcβ 1-3Fucα 1-O-Ser/Thr. Similarly, O-glucose can be elongated by Gxylt1 or Gxylt2, followed by Xxylt1, resulting in the trisaccharide Xylα 1- 3Xylα 1-3Glcβ 1-O-Ser. O-linked glycosylation is known to be essential for the function of Notch. In addition, previous work showed that CADASIL mutants in mouse Notch3 EGF repeats 1-5 did not affect addition of O-fucose to Notch EGF repeats, but elongation of O-fucose by Fringe was reduced. These results suggest CADASIL mutations may influence glycosylation of mouse Notch3. My project has been to analyze O-fucose and O-glucose modifications on wild type human Notch3 as a precursor to analyzing whether CADASIL mutations in the human protein alter their structure. Using mass spectral methods I have identified O-fucose and O- glucose glycans on the majority of predicted sites, and O-glucose was elongated to its trisaccharide form at most sites. However, glycosylation at several " non-canonical" sites were also identified. More work needs to be done to identify the remaining glycosylation sites on Notch3, and then the effects of CADASIL mutations on the efficiency of modification at individual sites can be addressed.