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Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation

Liang, Yan
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1343410954

Abstract Details

2012, Doctor of Philosophy (PhD), Ohio University, Molecular and Cellular Biology (Arts and Sciences).
Arabinogalactan-proteins (AGPs) are highly glycosylated hydroxyproline-rich glycoproteins (HRGPs) that are frequently characterized by the presence of the repetitive dipeptide motifs [Ala-Hyp] or [Hyp-Ala]. Hydroxyproline (Hyp) residues in the dipeptide motifs are sites for the attachment of arabinogalactan (AG) sugar side chains. Fucose residues are found in some dicot AGPs, and AGP fucosylation is developmentally regulated. AGP galactosyltransferase (GalT) and fucosyltransferase (FUT) activities were investigated in three ways in this study. First, an in vitro AGP GalT assay was developed, which used permeabilized microsomal membranes from tobacco (Nicotiana tobacum) or Arabidopsis (Arabidopsis thaliana) suspension-cultured cells as the enzyme source and UDP-[14C] Gal as the sugar donor. Two model AGP peptides, [Ala-Hyp]7 or [AO]7 and deglycosylated [Ala-Hyp]51 or d[AO]51, were used as substrate acceptors. Product analysis indicated that the [AO]7:GalT assay and the d[AO]51:GalT assay mainly detected GalT activities that added the first and the second Gal residues in the Hyp-AG side chain. Examination of the Hyp:GalT activity using various acceptor substrates, including two extensin sequences and a [AP]7 peptide, indicated this activity was specific for Hyp dipeptide motifs in AGPs. The [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. Second, five candidate AGP GALT genes (GALT1: At1g26810; GALT3: At3g06440; GALT4: At1g27120; GALT5: At1g74800; GALT6: At5g62620) were expressed in Pichia pastoris and tobacco suspension-cultured cells and tested for AGP GalT activity using the [AO]7:GalT and d[AO]51:GalT assay systems. Preliminary results indicate that heterologously expressed GalT3, GalT5 and GalT6 have AGP GalT activities. Furthermore, GalT3 was shown to be Golgi localized in a tobacco leaf expression system. Third, functions of the FUT4 and FUT6 genes were investigated using Arabidopsis fut4, fut6 and fut4/fut6 mutant plants. Biochemical analysis indicated that FUT4 was required for fucosylation of leaf AGPs, while both FUT4 and FUT6 contributed to fucosylation of root AGPs. In addition, glycome profiling indicated that fucosylated AGPs may regulate intermolecular interactions between AGPs and other wall components. Finally, a model of HRGP biosynthesis is proposed which highlights the glycosyltransferases involved in this process.
Allan Showalter, PhD (Advisor)
Ahmed Faik, PhD (Committee Member)
Marcia Kieliszewski, PhD (Committee Member)
Sarah Wyatt, PhD (Committee Member)
239 p.
Biochemistry; Biology; Cellular Biology; Molecular Biology; Physiology; Plant Biology
Arabinogalactan-proteins; galactosyltransferase; fucosyltransferase; hydroxyproline-rich glycoproteins; plant cell walls

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Citations

  • Liang, Y. (2012). Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1343410954

    APA Style (7th edition)

  • Liang, Yan. Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation. 2012. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1343410954.

    MLA Style (8th edition)

  • Liang, Yan. "Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation." Doctoral dissertation, Ohio University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1343410954

    Chicago Manual of Style (17th edition)

ohiou1343410954
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© 2012, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.