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

Abstract

Mucin-type -glycosylation is the most abundant type of-glycosylation. It is initiated by the members of the polypeptide -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-glycosylation. Here, we report that a small molecule, the plant flavonoid luteolin, selectively inhibits ppGalNAc-Ts and in cells. We found that luteolin inhibits ppGalNAc-T2 in a peptide/protein-competitive manner but not promiscuously ( via aggregation-based activity). X-ray structural analysis revealed that luteolin binds to the PP 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 H 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-GalNAc glycosylation and into the molecular mechanism of luteolin-mediated ppGalNAc-T inhibition.

The Multiplicity of Polypeptide GalNAc‐Transferase: Assays, Inhibitors, and Structures

“…The assay utilizes a tandem enzyme reaction catalyzed by pyruvate kinase (PK) and lactate dehydrogenase (LDH) to link the generation of UDP, the enzymatic product of GTs, with the consumption of nicotinamide adenine dinucleotide (NADH), which has an absorbance at 340 nm. The assay was not adopted to measure the activity of GalNAc‐T until we recently set up and optimized the assay conditions for purified GalNAc‐T2 (Figure A) . The advantage of this tandem enzyme‐coupling assay for GalNAc‐T2 is that it avoids the use of a radioactive substrate and special instruments for detection .…”

“…Also, the signal from the hydrolysis of UDP‐GalNAc rather than that from the activity of the transferring enzyme could theoretically interfere with the readout of the assay; however, the nonspecific hydrolysis activity seems negligible under optimal enzyme assay conditions for GalNAc‐Ts . Nevertheless, we could employ this method to perform a high‐throughput inhibitor screening campaign and to identify several micromolar inhibitors for GalNAc‐T2 …”

“…Moreover, the abundance of the substrate or product peak can be simultaneously quantified by measuring the fluorescence signals from the 5‐FAM fluorophore at an emission wavelength of 520 nm ( λ ex =492 nm). This assay has been and is still successfully used to study the activities of GalNAc‐Ts and their substrate specificities . However, owing to its low throughput, it has not been applied for GalNAc‐T inhibitor screening.…”

“…Furthermore, a few direct inhibitors have been designed and developed for GalNAc‐Ts, and most of them are not potent or bioactive in cells. In the following section, we itemized the available inhibitors for GalNAc‐Ts (Table ).…”

“…In the following section, we itemized the available inhibitors for GalNAc‐Ts (Table ). These inhibitors include a synthetic inhibitor for GalNAc‐T3, natural product inhibitors, and UDP‐GalNAc‐analogue‐based pan‐inhibitors …”