Characterization of Endoplasmic Reticulum-Localized UDP-d-Galactose: HydroxyprolineO-Galactosyltransferase Using Synthetic Peptide Substrates in Arabidopsis

Takuji Oka,Yoh-Ichi Shimma,Takehiko Yoko-O,Yoshifumi Jigami,Yoshiyuki Nomura,Fumie Saito,Ken Matsuoka

doi:10.1104/pp.109.146266

Abstract

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. An in vitro assay of HGT activity was established using chemically synthesized fluorescent peptides as acceptor substrates and extracts from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of crude enzyme. The galactose residue transferred to the peptide could be detected by high-performance liquid chromatography and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry analyses. HGT required a divalent cation of manganese 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 degrees C. The favorable substrates for the activity seemed to be peptides containing two alternating imino acid residues including at least one acceptor Hyp residue, although a peptide with single Hyp residue without any other imino acids also functioned as a substrate. The results of sucrose density gradient centrifugation revealed that the cellular localization of HGT activity is identical to those of endoplasmic reticulum markers such as Sec61 and Bip, indicating that HGT is predominantly localized to the endoplasmic reticulum. To our knowledge, this is the first characterization of HGT, and the data provide evidence that arabinogalactan biosynthesis occurs in the protein transport pathway.

Highlights

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway
We report a novel assay for Hyp O-galactosyltransferase (HGT) activity based on the use of endoplasmic reticulum (ER)-enriched cell lysates extracted from Arabidopsis (Arabidopsis thaliana) T87 cells as a source of the enzyme and chemically synthesized fluorescent peptides as enzyme substrates
The substrate peptides were chemically synthesized with two modifications at the N terminus; namely, addition of fluorescein isothiocyanate (FITC) to facilitate detection of the peptide and addition of g-aminobutyric acid (GABA) as a spacer (Table I)

Summary

Introduction

We characterized peptidyl hydroxyproline (Hyp) O-galactosyltransferase (HGT), which is the initial enzyme in the arabinogalactan biosynthetic pathway. The results of sucrose density gradient centrifugation revealed that the cellular localization of HGT activity is identical to those of endoplasmic reticulum markers such as Sec and Bip, indicating that HGT is predominantly localized to the endoplasmic reticulum To our knowledge, this is the first characterization of HGT, and the data provide evidence that arabinogalactan biosynthesis occurs in the protein transport pathway. Shpak et al described a novel strategy to elucidate O-glycosylation of AGPs via introduction of synthetic genes encoding a protein substrate of glycosyltransferases into plant cells (Shpak et al, 1999; Estevez et al, 2006) This strategy provided good evidence for the substrate specificities of Hyp O-galactosyltransferase (HGT). Properties of the enzyme and the usefulness of our assay for various studies are discussed

Methods

Results

Conclusion