How does GDP‐fucose get into the Endoplasmic Reticulum for O‐fucosylation of EGF Repeats and TSRs?

Abstract

Fucose is a common monosaccharide found in glycan chains of all mammalian cells. Fucose is transferred from GDP‐fucose to glycoproteins by fucosyltransferases, resulting in terminal fucosylation of N‐linked glycans or mucin‐type O‐glycans, or direct modification of serine or threonine residues by Protein O‐fucosyltransferases (POFUT) 1 or 2. Defects in fucosylation can lead to developmental disorders or cancer. In particular, POFUT1 adds O‐fucose to Epidermal Growth Factor‐like (EGF) repeats of Notch receptors, while POFUT2 adds O‐fucose to Thrombospondin type 1 Repeats (TSRs) found in multiple cell‐surface and secreted proteins, including the A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) family of extracellular proteases. Elimination of Pofut1 or Pofut2 results in early embryonic lethality in mice, indicating these two fucosylation pathways play essential roles in mouse development. Both POFUT1 and POFUT2 are located in the endoplasmic reticulum (ER), while fucosyltransferases involved in terminal fucosyaltion are localized in the trans‐Golgi. GDP‐fucose is synthesized in the cytoplasm either through a de novo or salvage pathway, and the GDP‐fucose is then transported into the lumen of the ER or Golgi for use by the fucosyltransferases. Currently two GDP‐fucose transporters, SLC35C1 and SLC35C2, have been identified, both of which are localized to the Golgi. It is unknown how GDP‐fucose is transported into the ER where Notch receptors and ADAMTS proteins are fucosylated. In order to uncover where the GDP‐fucose for POFUT1 and POFUT2 comes from, we have generated CRISPR‐Cas9 mutant cell lines in which fucosylation is impaired in two ways: 1. Inactivating GDP‐fucose biosynthetic enzymes from the salvage or de novo pathways; 2. Inactivating the GDP‐fucose transporters. HEK293T cells have proven to be an excellent model for fucosylation studies. All the fucose biosynthesis genes are normally expressed, and impaired fucosylation can be expected in knockout mutant cell lines. Using CRISPR‐Cas9 technology, the following genes have been knocked out: GMDS and TSTA3 of the de novo pathway, FUK and GFPP of the salvage pathway, and the GDP‐fucose transporters SLC35C1 and SLC35C2. O‐Fucosylation of EGF repeats and TSRs and terminal fucosylation of N‐linked glycans is being analyzed using Mass Spectrometry and lectin binding. Notch function is also being evaluated. These results should reveal whether a preferred biosynthetic pathway or transporter for different forms of fucosylation exists.Support or Funding InformationSupported by NIH grant GM061126.