Abstract
BackgroundArabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood.ResultsWe have identified a novel enzyme in the biosynthesis of the glycan moiety of arabinogalactan proteins. The At1g08280 (AtGALT29A) from Arabidopsis thaliana encodes a putative glycosyltransferase (GT), which belongs to the Carbohydrate Active Enzyme family GT29. AtGALT29A co-expresses with other arabinogalactan GTs, AtGALT31A and AtGLCAT14A. The recombinant AtGALT29A expressed in Nicotiana benthamiana demonstrated a galactosyltransferase activity, transferring galactose from UDP-galactose to a mixture of various oligosaccharides derived from arabinogalactan proteins. The galactose-incorporated products were analyzed using structure-specific hydrolases indicating that the recombinant AtGALT29A possesses β-1,6-galactosyltransferase activity, elongating β-1,6-galactan side chains and forming 6-Gal branches on the β-1,3-galactan main chain of arabinogalactan proteins. The fluorescence tagged AtGALT29A expressed in N. benthamiana was localized to Golgi stacks where it interacted with AtGALT31A as indicated by Förster resonance energy transfer. Biochemically, the enzyme complex containing AtGALT31A and AtGALT29A could be co-immunoprecipitated and the isolated protein complex exhibited increased level of β-1,6-galactosyltransferase activities compared to AtGALT29A alone.ConclusionsAtGALT29A is a β-1,6-galactosyltransferase and can interact with AtGALT31A. The complex can work cooperatively to enhance the activities of adding galactose residues 6-linked to β-1,6-galactan and to β-1,3-galactan. The results provide new knowledge of the glycosylation process of arabinogalactan proteins and the functional significance of protein-protein interactions among O-glycosylation enzymes.
Highlights
Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation
The transcript levels are generally low in Arabidopsis throughout development, but higher during seed maturation and root development, and the gene is co-expressed with AtGALT31A [17] and AtGLCAT14A [18], which were recently identified as possessing galactosyltransferase and glucuronosyltransferase activity, respectively, involved in the glycosylation of type II AGs (GeneCAT, http://genecat.mpg.de) [23] (Additional file 1: Figure S1)
Recombinant protein encoded by At1g08280 showed galactosyltransferase activity towards type II arabinogalactan acceptors For biochemical characterization, the full-length At1g08280 construct harboring N-terminal HA tag was expressed in N. benthamiana and affinity purified using monoclonal anti-HA-antibody conjugated to agarose
Summary
Arabinogalactan proteins are abundant proteoglycans present on cell surfaces of plants and involved in many cellular processes, including somatic embryogenesis, cell-cell communication and cell elongation. Arabinogalactan proteins consist mainly of glycan, which is synthesized by post-translational modification of proteins in the secretory pathway. Importance of the variations in the glycan moiety of arabinogalactan proteins for their functions has been implicated, but its biosynthetic process is poorly understood. Arabinogalactan proteins (AGPs) are an abundant class of proteoglycans in plant cell walls and are implicated in the control of cell proliferation and morphogenesis [1]. Subtle differences in the glycan structure of AGPs may function as markers used in coordinating developmental processes in plants. The glycans of AGPs originate by post-translational modification of protein backbones catalyzed by glycosyltransferases (GTs) in the secretory pathway. The glycan structure of AGPs is heterogeneous, but commonly composed of a β-1,3-linked galactan backbone with substitution of the side chains at O6 positions (type II AG). It is anticipated that more than 10 functionally distinct GTs are required to build the AGP glycans, and so far fucosyltransferases (AtFUT4, AtFUT6) [15], galactosyltransferases (AtGALT2 [16] and AtGALT31A [17]), and a glucuronosyltransferase (AtGLCAT14A) [18] have been characterized
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