The small molecule luteolin inhibits N-acetyl-α-galactosaminyltransferases and reduces mucin-type O-glycosylation of amyloid precursor protein.

Mucin-type O-glycosylation is the most abundant type of O-glycosylation. It is initiated by the members of the polypeptide N-acetyl-α-galactosaminyltransferase (ppGalNAc-T) family and closely associated with both physiological and pathological conditions, such as coronary artery disease or Alzheimer's disease. The lack of direct and selective inhibitors of ppGalNAc-Ts has largely impeded research progress in understanding the molecular events in mucin-type O-glycosylation. Here, we report that a small molecule, the plant flavonoid luteolin, selectively inhibits ppGalNAc-Ts in vitro and in cells. We found that luteolin inhibits ppGalNAc-T2 in a peptide/protein-competitive manner but not promiscuously (e.g. via aggregation-based activity). X-ray structural analysis revealed that luteolin binds to the PXP motif-binding site found in most protein substrates, which was further validated by comparing the interactions of luteolin with wild-type enzyme and with mutants using 1H NMR-based binding experiments. Functional studies disclosed that luteolin at least partially reduced production of β-amyloid protein by selectively inhibiting the activity of ppGalNAc-T isoforms. In conclusion, our study provides key structural and functional details on luteolin inhibiting ppGalNAc-T activity, opening up the way for further optimization of more potent and specific ppGalNAc-T inhibitors. Moreover, our findings may inform future investigations into site-specific O-GalNAc glycosylation and into the molecular mechanism of luteolin-mediated ppGalNAc-T inhibition.