Abstract
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 [(14)C]Gal from UDP-[(14)C]Gal into the [AO](7) and d[AO](51) acceptors was observed following HPLC fractionation of the reaction products. Hyp-[(14)C]Gal monosaccharide and Hyp-[(14)C]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 SO(4) 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.
Highlights
Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline (Hyp)-rich glycoproteins that are frequently characterized by the presence of [Alanine-Hyp] ([AO]) repetitive units
An in vitro GalT assay system was developed with UDP-[14C]Gal as the sugar donor and permeablized microsomal membranes from tobacco or Arabidopsis suspension-cultured cells as the enzyme resource
The d[AO]51 peptide was obtained from the [AO]51EGFP fusion protein expressed in transgenic tobacco BY-2 cells; the EGFP tag was removed by trypsin digestion, and the AG side chains were removed by deglycosylation with hydrogen fluoride
Summary
Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline (Hyp)-rich glycoproteins that are frequently characterized by the presence of [Alanine-Hyp] ([AO]) repetitive units. 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. Arabinogalactan-proteins (AGPs) are highly glycosylated Hyp-rich glycoproteins (HRGPs) implicated in many physiological processes, including plant somatic embryogenesis, programmed cell death, wound responses, and hormone signaling pathways (Seifert and Roberts, 2007; Ellis et al, 2010). AGPs are defined by the presence of arabinogalactan (AG) polysaccharides and reside mainly at the plasma membrane-cell wall interface and in plant exudates (Langan and Nothnagel, 1997; Oxley and Bacic, 1999; Sherrier et al, 1999; Svetek et al, 1999; Lamport et al, 2006) These AG polysaccharides are added via O-glycosylation, which is widespread in plants and includes monogalactosylation of Ser residues and extensive modification of Hyp, which ranges from addition of oligoarabinosides to AG polysaccharide addition. This work was achieved by developing an in vitro AGP GalT assay using synthetic and transgenically produced AGP peptides as acceptor substrates, UDP[14C]Gal as the sugar donor, and permeabilized microsomal membranes from tobacco BY2 and Arabidopsis suspension-cultured cells
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