Characterization of endoplasmic reticulum-localized UDP-D-galactose

Hydroxyproline O-galactosyltransferase using synthetic peptide substrates in arabidopsis

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

Research output: Contribution to journalArticle

23 Citations (Scopus)

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°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.

Original languageEnglish
Pages (from-to)332-340
Number of pages9
JournalPlant physiology
Volume152
Issue number1
DOIs
Publication statusPublished - Jan 1 2010

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Uridine Diphosphate Galactose
galactosyltransferases
Galactosyltransferases
synthetic peptides
hydroxyproline
Hydroxyproline
Galactose
Arabidopsis
Endoplasmic Reticulum
galactose
endoplasmic reticulum
Peptides
Imino Acids
peptides
arabinogalactans
Density Gradient Centrifugation
protein transport
matrix-assisted laser desorption-ionization mass spectrometry
Biosynthetic Pathways
Divalent Cations

All Science Journal Classification (ASJC) codes

  • Plant Science
  • Genetics
  • Physiology

Cite this

Characterization of endoplasmic reticulum-localized UDP-D-galactose : Hydroxyproline O-galactosyltransferase using synthetic peptide substrates in arabidopsis. / Oka, Takuji; Saito, Fumie; Shimma, Yoh ichi; Yoko-o, Takehiko; Nomura, Yoshiyuki; Matsuoka, Ken; Jigami, Yoshifumi.

In: Plant physiology, Vol. 152, No. 1, 01.01.2010, p. 332-340.

Research output: Contribution to journalArticle

Oka, Takuji ; Saito, Fumie ; Shimma, Yoh ichi ; Yoko-o, Takehiko ; Nomura, Yoshiyuki ; Matsuoka, Ken ; Jigami, Yoshifumi. / Characterization of endoplasmic reticulum-localized UDP-D-galactose : Hydroxyproline O-galactosyltransferase using synthetic peptide substrates in arabidopsis. In: Plant physiology. 2010 ; Vol. 152, No. 1. pp. 332-340.
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