title: Functional identification of a hydroxyproline-o-galactosyltransferase specific for arabinogalactan protein biosynthesis in Arabidopsis.
abstract: Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [(14)C]Gal from UDP-[(14)C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)7 and anhydrous hydrogen fluoride-deglycosylated d(AO)51. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [(14)C]Gal to (AO)7, and the resulting product co-eluted with (AO)7 by reverse-phase HPLC. Acid hydrolysis of the [(14)C]Gal-(AO)7 product released (14)C-radiolabel as Gal only. Base hydrolysis of the [(14)C]Gal-(AO)7 product released a (14)C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg(2+) or Mn(2+) for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.
authors: Department of Environmental and Plant Biology, Molecular and Cellular Biology Program, Ohio University, Athens, Ohio 45701-2979.
authors: Debarati
authors: D
authors: Basu
authors: Department of Environmental and Plant Biology, Molecular and Cellular Biology Program, Ohio University, Athens, Ohio 45701-2979.
authors: Yan
authors: Y
authors: Liang
authors: Department of Environmental and Plant Biology, Molecular and Cellular Biology Program, Ohio University, Athens, Ohio 45701-2979.
authors: Xiao
authors: X
authors: Liu
authors: Department of Chemistry and Biochemistry, Molecular and Cellular Biology Program, Ohio University, Athens, Ohio 45701-2979.
authors: Klaus
authors: K
authors: Himmeldirk
authors: Department of Environmental and Plant Biology, Molecular and Cellular Biology Program, Ohio University, Athens, Ohio 45701-2979.
authors: Ahmed
authors: A
authors: Faik
authors: Department of Chemistry and Biochemistry, Molecular and Cellular Biology Program, Ohio University, Athens, Ohio 45701-2979.
authors: Marcia
authors: M
authors: Kieliszewski
authors: Department of Chemistry and Biochemistry, Molecular and Cellular Biology Program, Ohio University, Athens, Ohio 45701-2979.
authors: Michael
authors: M
authors: Held
authors: Department of Environmental and Plant Biology, Molecular and Cellular Biology Program, Ohio University, Athens, Ohio 45701-2979. Electronic address: showalte@ohio.edu.
authors: Allan M
authors: AM
authors: Showalter
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism / Q000502:physiology*; D002384:Catalysis; D002473:Cell Wall / Q000378:metabolism; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular; D005685:Galactans / Q000737:chemistry* / Q000378:metabolism; D005700:Galactosyltransferases / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D006909:Hydroxyproline / Q000737:chemistry; D015151:Immunoblotting / Q000379:methods; D018613:Microscopy, Confocal / Q000379:methods; D008861:Microsomes / Q000378:metabolism; D008968:Molecular Conformation; D009154:Mutation; D010843:Pichia / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D005685:Galactans; D005700:Galactosyltransferases; C548154:UDP-D-galactose hydroxyproline O-galactosyltransferase, Arabidopsis; D006909:Hydroxyproline; C005653:arabinogalactan