期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 288, 期 14, 页码 10132-10143出版社
ELSEVIER
DOI: 10.1074/jbc.M112.432609
关键词
-
资金
- Ohio Plant Biotechnology Consortium [020340004090 GR0017687.01]
- National Science Foundation [0918661]
- Direct For Biological Sciences
- Division Of Integrative Organismal Systems [0918661] Funding Source: National Science Foundation
- Division Of Integrative Organismal Systems
- Direct For Biological Sciences [1145887] Funding Source: National Science Foundation
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 [C-14] Gal from UDP-[C-14] 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 [C-14] Gal to (AO)(7), and the resulting product co-eluted with (AO)(7) by reverse-phase HPLC. Acid hydrolysis of the [C-14]Gal-(AO)(7) product released C-14-radiolabel as Gal only. Base hydrolysis of the [C-14] Gal-(AO)(7) product released a C-14-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 Mg2+ or Mn2+ 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 beta-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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据