Abstract
We characterized peptidyl hydroxyproline O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan (AG) biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis thaliana T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by HPLC and MALDI-TOF-MS analyses. HGT required Mn2+ for maximal activity and consumed UDP-D-galactose as a sugar donor. HGT exhibited an optimal pH range of pH 7.0 to 8.0 and an optimal temperature of 35 degree. The favorable substrates for the activity seemed peptides containing two alternating imino acid residues including at least one acceptor hydroxyproline (HYP) residue, although a peptide with single HYP residue without any other imino acids also functioned as a substrate. The cellular localization of HGT activity is identical to those of ER markers, indicating that HGT is predominantly localized to the ER. This is the first characterization of HGT and the data provide evidence that AG biosynthesis occurs in the protein transport pathway.
