RRC ID 30737
Author Basu D, Liang Y, Liu X, Himmeldirk K, Faik A, Kieliszewski M, Held M, Showalter AM.
Title Functional identification of a hydroxyproline-o-galactosyltransferase specific for arabinogalactan protein biosynthesis in Arabidopsis.
Journal J. Biol. Chem.
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.
Volume 288(14)
Pages 10132-43
Published 2013-4-5
DOI 10.1074/jbc.M112.432609
PII M112.432609
PMID 23430255
PMC PMC3617256
MeSH Arabidopsis / metabolism* Arabidopsis Proteins / metabolism Arabidopsis Proteins / physiology* Catalysis Cell Wall / metabolism Chromatography, Ion Exchange / methods Cloning, Molecular Galactans / chemistry* Galactans / metabolism Galactosyltransferases / chemistry Galactosyltransferases / metabolism Galactosyltransferases / physiology* Gene Expression Regulation, Enzymologic* Gene Expression Regulation, Plant* Glycosylation Hydroxyproline / chemistry Immunoblotting / methods Microscopy, Confocal / methods Microsomes / metabolism Molecular Conformation Mutation Pichia / metabolism Plant Leaves / metabolism Substrate Specificity
IF 4.106
Times Cited 31
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
Arabidopsis / Cultured plant cells, genes pda01627 pda09678 pda08691 pda11283