Protein O-fucosyltransferase (POFUT) enzymes mediate direct O-fucosylation on secreted and membrane-associated proteins such as thrombospondin-1 and Notch-11,2. Dysregulation of O-fucosylation has been linked to numerous cancers and developmental disorders3. POFUTs play a key role in modulating the secretion of their substrates, by O-fucosylating specific substrate protein domains only when correctly folded, and as such are thought to be involved in non-canonical protein quality control4. However, as O-glycosylation is critically understudied, the true diversity of proteins regulated by O-fucosylation is largely uncharacterised5.
We recently identified the human FUT10/FUT11 enzymes as responsible for protein O-fucosylation of Elastin Microfibril Interface (EMI) domains6. Here, we combined Alphafold2 protein structural prediction and the structural alignment tool Foldseek to identify new substrates for FUT10/FUT11. Each human EMI-domain was analysed in Foldseek using an iterative approach to detect structurally homologous protein domains. Numerous proteins with EMI-like domains were identified, including microfibrillar-associated protein 2/5 (MFAP2/MFAP5), which have key roles in signalling within the extracellular matrix. The structure of the EMI-like domain is highly conserved in these proteins despite low primary sequence homology. As such, we hypothesised they may be recognised by the structurally-specific FUT10/FUT11 enzymes. O-fucosylation within these new EMI-like domains was confirmed at high stoichiometry in mouse tissues. To identify the enzyme modifying these proteins, HEK293 cells were transiently transfected with MFAP2/5 with or without FUT10/FUT11 to quantify interactions and O-fucosylation. Interestingly, O-fucosylation of MFAP2/MFAP5 is only observed in cells co-transfected with FUT10, but not with FUT11, a selectivity that has not been seen for EMI domains. To functionally characterise O-fucosylation of these EMI-like domains, we performed secretion assays comparing wild-type MFAP2 with an alanine mutant at the modified residue, preventing O-fucosylation. This mutant showed >50% loss in secretion, indicating that FUT10 is required for proper secretion of MFAP2. Future work will investigate the role of this O-fucosylation through FUT10/11 knockout mice, and streamline this workflow for application to other enzymes. A methodology for rapid identification of new enzyme substrates would allow a deeper understanding of the diverse roles enzymes may play in different contexts and broaden our knowledge of the human interactome.