Structure of human POFUT1, its requirement in ligand-independent oncogenic Notch signaling, and functional effects of Dowling-Degos mutations

Abstract

Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signaling are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1-/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.

Mechanistic insights from plant heteromannan synthesis in yeast

“…S5B). There, the R240 residue is indispensable for HsPoFUT1 catalytic activity, and D340 is within hydrogen-bonding distance of the guanine ring on GDPfucose (28). Moreover, disulfide bridges contribute to the formation of the PoFUT1 sugar-binding site (29), with the C274 amino acid of AtMSR1 putatively involved in this process (SI Appendix, Fig.…”

“…Therefore, specific MSR cofactors are likely involved in the production of distinct types of HM by modulating CSLA activity. Plant MSR cofactors have sequence homology to mammalian PoFUT1 enzymes, which decorate proteins with O-glycan groups that are essential for cell development (28). Mutagenesis of three AtMSR1 conserved amino acids, which are involved in nucleotide sugar binding by PoFUT1 enzymes (28,29), significantly reduced 4-Glc as well as 4-Man incorporation by AtCSLA2 into AKI polymers ( Fig.…”

A perspective on structural and mechanistic aspects of proteinO-fucosylation

“…For both enzymes, most of the residues involved in interaction with GDP-fucose are conserved. The essential residues Arg240 HsPoFUT1 /Arg294 HsPoFUT2 interact with the -phosphate of GDP-fucose through hydrogen-bonding and electrostatic topical reviews interactions, which are conserved in other fucosyltransferases (Martinez-Duncker et al, 2003;Okajima et al, 2005;Lira-Navarrete et al, 2011;Chen et al, 2012;McMillan et al, 2017). The GDP moiety is tethered by additional interactions with Asn46/Asn57, His238/His292, Asp340/Asp371, Ser356/Ser387, Ser357/Thr388 and Phe358/Phe389 of HsPoFUT1 and HsPoFUT2, respectively (Fig.…”

A phenotype combining hidradenitis suppurativa with Dowling-Degos disease caused by a founder mutation in PSENEN

“…8,9 Decreased Notch activity is associated with mutations in POFUT1. 31 PSENEN and POGLUT1 expressions were downregulated in patient keratinocytes. In addition, abnormal expression of known effector genes of this pathway, HEYL, HES1 and HES5, was also observed.…”