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

Abstract

Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline (Hyp)-rich glycoproteins that are frequently characterized by the presence of [Alanine-Hyp] GalT activities were localized to the endomembrane system of Arabidopsis suspension-cultured cells following sucrose density gradient centrifugation. The in vitro assay reported here to detect GalT activities using AGP peptide and glycopeptide acceptor substrates provides a useful tool for the identification and verification of AGP-specific GalT proteins/genes and an entry point for elucidation of arabinogalactan biosynthesis for AGPs.

Engineering Mammalian Mucin-type O-Glycosylation in Plants

“…P4Hs are membrane-bound type II proteins found in the ER and extending into the Golgi apparatus (16). Thirteen putative P4Hs have been identified in Arabidopsis (58). In this study a minor amount of hydroxylation of Pro 11 of the MUC1 tandem repeat was observed.…”

Arabinogalactan Proteins in Arabidopsis thaliana Pollen Development

“…AGP galactosyltransferase (GalT) activities in tobacco and Arabidopsis microsomal membranes were studied with an in vitro GalT reaction system. This in vitro assay reported to detect GalT activities using AGP peptide and glycopeptide acceptor substrates provides a useful tool for the identification and verification of AGPspecific GalT proteins/genes and an entry point for elucidation of arabinogalactan biosynthesis for AGPs (Liang et al, 2010).…”

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis

“…In other cases, the lack of a robust and reproducible enzyme assay to validate their function presents another challenge. Liang et al (13) proposed that as many as 15 different GTs may be involved in the biosynthesis of AGPs, of which only two GTs, specifically two fucosyltransferases, have been successfully characterized and shown to add terminal fucose (Fuc) residues on AGPs (14). Another candidate gene, encoding a putative transferase, has been recently demonstrated to transfer Gal to an O-methylated Gal-␤-(1,3)-Gal disaccharide acceptor, an analog of the ␤-(1,3)-galactan chains found in AGPs (8).…”

“…Thus, it is hypothesized that the GTs responsible for adding the first sugar to the protein core of mammalian proteoglycans should be similar to the GTs responsible for adding the first sugar to the AGP protein backbone. Recently, Liang et al (13) and Oka et al (20) reported on novel in vitro assays using synthetic AGP peptides for detecting Hyp-O-GALT activities in Arabidopsis microsomal membranes. Using the protocol published by Liang et al (13), we report here on the identification and functional characterization of an Arabidopsis At4g21060 gene (named AtGALT2) that encodes a ␤-GALT involved in the biosynthesis of the glycan chain of AGPs.…”