Two NOTCH1 O-fucose sites have opposing functions in mouse retinal angiogenesis.

Previous in vitro studies demonstrated that Fringe glycosylation of the NOTCH1 extracellular domain at O-fucose residues in Epidermal Growth Factor-like Repeats (EGFs) 6 and 8 is a significant contributor to suppression of NOTCH1 activation by JAG1 or enhancement of NOTCH1 activation by DLL1, respectively. In this study we sought to evaluate the significance of these glycosylation sites in a mammalian model by generating two C57BL/6 J mouse lines carrying NOTCH1 point mutations which eliminate O-fucosylation and Fringe activity at EGFs 6 (T232V) or 8 (T311V). We assessed changes to morphology during retinal angiogenesis, a process in which expression of Notch1, Jag1, Dll4, Lfng, Mfng, and Rfng genes coordinate cell-fate decisions to grow vessel networks. In the EGF6 O-fucose mutant (6f/6f) retinas we observed reduced vessel density and branching, suggesting that this mutant is a Notch1 hypermorph. This finding agrees with prior cell-based studies showing that the 6f mutation increased JAG1 activation of NOTCH1 during co-expression with inhibitory Fringes. Although we predicted that the EGF8 O-fucose mutant (8f/8f) would not complete embryonic development due to the direct involvement of the O-fucose in engaging ligand, the 8f/8f mice were viable and fertile. In the 8f/8f retina we measured increased vessel density consistent with established Notch1 hypomorphs. Overall, our data supports the importance of NOTCH1 O-fucose residues for pathway function and confirms that single O-glycan sites are rich in signaling instructions for mammalian development.© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Citation

Rachel K LoPilato, Heike Kroeger, Sneha K Mohan, James D Lauderdale, Neil Grimsey, Robert S Haltiwanger. Two NOTCH1 O-fucose sites have opposing functions in mouse retinal angiogenesis. Glycobiology. 2023 Jun 17

PMID: 37329502

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