GGT Enzymatic Activity Assay

GGT activity was measured similarly as previously described^{12 (link)}. The assay was performed in reaction buffer (100 mM HEPES buffer pH 8.0, 150 mM NaCl) at 37 °C for 1 h with 1 μM R699 enzyme, 100 μM MnCl₂, 200 μM UDP-glucose (MilliporeSigma, St. Louis, MO), 1 mM dithiothreitol and 1.75 mM galactosyl hydroxylysine (Gal-Hyl, Cayman Chemical, Ann Arbor, MI) or 2 μM deglucosylated collagen IV. Deglucosylated collagen IV was generated using a glycosidase PGGHG as previously described^{12 (link)}. GGT activity was measured by detecting UDP production with an ATP–based luciferase assay (UDP-Glo™ Glycosyltransferase Assay, Promega, Madison, WI) according to manufacturers' instructions. Experiments were performed in triplicate from distinct samples, and an unpaired t-test was used to compare the enzymatic activity of different samples. The glucosylation of galactosyl hydroxylysine was further confirmed by mass spectrometry.