Domain-specific protein O-fucosylation plays critical roles in modulating the biological functions of numerous cell surface or secreted proteins. Previously, only Protein O-fucosyltransferase (POFUT) 1 mediated O-fucosylation of epidermal growth factor-like (EGF) domains and POFUT2-mediated O-fucosylation of thrombospondin type 1 repeats (TSRs) was known. Using high sensitivity glycoproteomics, we recently identified a novel type of O-fucosylation on elastin microfibril interface (EMI) domains. We determined that the enzymes FUT10 and FUT11 are previously uncharacterized POFUTs responsible for the O-fucosylation of EMI domains. Like POFUT1/2, FUT10/11 function in the endoplasmic reticulum, require folded domain structures for modification and participate in a non-canonical endoplasmic reticulum quality control pathway for EMI domain-containing protein secretion.
To identify additional FUT10/11 substrates, we used protein 3D structural similarity analysis with Foldseek to identify other EMI-like domain structures in the human proteome. We identified two elastin microfibril-associated proteins (MFAP-2/5) as having strong structural similarity and being O-fucosylated in human tissues. We have demonstrated that these proteins bind FUT10/11 strongly, but that unusually they are only O-fucosylated by FUT10, but not FUT11. This is the first time that a bias in the substrate preference for these fucosyltransferase enzymes has been observed. We have shown that similar to EMI domains, O-fucosylation of MFAP-2/5 is important for secretion. MFAP-2/5 play an important role in the extracellular matrix, where they control TGF-β signalling events critical for processes such as blood vessel formation. Our current work is investigating these functions of MFAP-2/5 using FUT10 knockout mice. Our discovery that MFAP-2/5 are substrates of FUT10 suggests there are likely many more substrates that need to be identified, to fully understand all the biological processes downstream of FUT10/11.