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

Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline (Hyp)-rich glycoproteins that are frequently characterized by the presence of [Alanine-Hyp] ([AO]) repetitive units. AGP galactosyltransferase (GalT) activities in tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) microsomal membranes were studied here with an in vitro GalT reaction system, which used acceptor substrates composed of [AO] repetitive units, specifically, a chemically synthesized [AO](7) acceptor and a transgenically produced and deglycosylated d[AO](51) acceptor. Incorporation of [C-14] Gal from UDP-[C-14]Gal into the [AO](7) and d[AO](51) acceptors was observed following HPLC fractionation of the reaction products. Hyp-[C-14]Gal monosaccharide and Hyp-[C-14] Gal disaccharide were identified in the base hydrolysates of the GalT reaction products, indicating the presence of two distinct GalT activities for the addition of the first and second Gal residues to the [AO] peptide in both tobacco and Arabidopsis. Examination of the Arabidopsis Hyp: GalT activity using various acceptor substrates, including two extensin sequences containing SO4 modules and a [AP](7) peptide, indicated this activity was specific for peptidyl Hyp in AGP sequences. Mass spectrometry analysis demonstrated that only one Gal was added per peptide molecule to the C-terminal or penultimate Hyp residue of the [AO](7) peptide. In addition, [AO](7) : GalT and d[AO](51):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.