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

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.

Identification and Characterization of in Vitro Galactosyltransferase Activities Involved in Arabinogalactan-Protein Glycosylation in Tobacco and Arabidopsis

“…S1) (Fig. 4) The Arabidopsis Hyp:GalT activity observed here is similar to that reported by Oka et al (2010) with respect to its properties (pH optimum, temperature optimum, requirement for Mn 2+ , and specificity for UDP-Gal; Fig. 5, Table I) and localization to the endomembrane system (Fig.…”

“…Protein N-glycosylation is initiated in ER but completed in Golgi apparatus. Oka et al (2010) proposed a hypothetical model for AG biosynthesis in which the first Gal residue is added to peptidyl Hyp in an AGP backbone by GalT in the ER with further AG side chain elongation occurring in the Golgi apparatus. The subcellular localization of the Hyp:GalT and Gal:GalT activities here is consistent with this model in that these enzyme activities were localized in the endomembrane system (Fig.…”

“…A bioinformatics study looking for Arabidopsis homologs to mammalian b-(1,3)GalTs identified 20 putative Arabidopsis b-(1,3)GalTs (Qu et al, 2008), one of which was previously identified as a b-(1,3)GalT involved in the biosynthesis of protein-bound N-linked oligosaccharide (N-glycan; Strasser et al, 2007). Additionally, Oka et al (2010) used an in vitro assay system to detect and localize Hyp:GalT activity in the endoplasmic reticulum (ER) of Arabidopsis using a chemically synthesized AGP peptide and variants thereof conjugated to fluorescein isothiocyanate via a g-aminobutyric acid as acceptor substrates. Finally, Wu et al (2010) identified and characterized two a-(1,2)fucosyltransferases encoded by AtFUT4 and AtFUT6 in Arabidopsis that are specific to AGPs.…”

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

“…, causing migration to heavier density portions in the gradient (Oka et al, 2010;Wulfetange et al, 2011). CLH1 distribution shifted from lighter portions (fractions 5-13) in the absence of Mg 2+ to heavier portions (fractions 11-17) of the Suc gradient in the presence of Mg…”

An Arabidopsis Cell Wall Proteoglycan Consists of Pectin and Arabinoxylan Covalently Linked to an Arabinogalactan Protein

“…Pectin and xylan synthesis occurs in the Golgi (Mohnen, 2008;Brown et al, 2011), and AGP synthesis initiates in the endoplasmic reticulum and continues in the Golgi (Oka et al, 2010;Wu et al, 2010). Thus, one possible mechanism for APAP1 biosynthesis is the addition of glycans onto AGP57C by either the consecutive addition of one residue at a time or by block addition, possibly occurring within the Golgi apparatus (Atmodjo et al, 2011(Atmodjo et al, , 2013.…”