Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation A dissertation presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Doctor of Philosophy Yan Liang August 2012 © 2012 Yan Liang. All Rights Reserved. 2 This dissertation titled Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation by YAN LIANG has been approved for the Department of Environmental and Plant Biology and the College of Arts and Sciences by Allan M. Showalter Professor of Environmental and Plant Biology Howard Dewald Interim Dean, College of Arts and Sciences 3 ABSTRACT LIANG, YAN, Ph.D., August 2012, Molecular and Cellular Biology Identification and Characterization of Galactosyltransferases and Fucosyltransferases Involved in Arabinogalactan-Protein Glycosylation Director of Dissertation: (Allan M. Showalter) 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 14 UDP-[ C] 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 4 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. Approved: _____________________________________________________________ Allan M. Showalter Professor of Environmental and Plant Biology 5 ACKNOWLEDGMENTS First and foremost, I am thankful to my advisor, Dr. Allan Showalter, the one who provided guidance throughout my PhD study; the one who offered the research assistant fellowship to allow me to concentrate on my research; the one who encouraged and trusted me in trying out my own ideas in experiments. I especially thank Dr. Ahmed Faik, who shared his expertise in enzyme biochemistry, his ideas of experimental design and his lab equipment, which were indispensable for the progress of my project. I also thank Dr. Marcia Kieliszewski, who is a critical thinker in science and at the same time impressed me with the feeling that doing science is an enjoyable process. I am grateful to Dr. Sarah Wyatt, not only for the information she taught me, but also for her guidance and help in becoming socialized in the academic society. I thank Dr. Wen-liang Xu for his contribution to the RNA analysis and HPAEC analysis in the FUT project and Ms. Alexandra Venetos and Ms. Rebecca Vondrell for their help in phenotypic analysis of the fut mutants. Mr. Xiao Liu and Mr. Wuda Wang helped with the bacterium colony screening in the expression construct cloning of GALT genes. Dr. Wei Zeng tutored me in the techniques of microsomal protein extraction, making sucrose gradient density, HPAEC and MS analysis and Pichia protein expression. Dr. Li Tan provided the d[AO]51 peptide substrate and helped to solve the technical problems with the RP-HPLC system. Ms. Debarati Basu helped with the GC/MS analysis and shared her experience in Pichia protein expression and activity tests. Dr. Michael Held helped to set up the tobacco leaf expression system and advised on the use of the confocal microscope. Dr. Betsy Briju, Ms. Laura Cristea and Mr. Vijayanand Nadella 6 provided suggestions in tobacco BY2 transformation. Mr. Yuning Chen and Mr. Dening Ye helped me with the liquid chromatography techniques. Dr. Glen Jackson and Dr. Hao Chen assisted with the ESI-MS/MS analysis. Dr. Jie Yang and Dr. Yizhu Zhang provided many helpful suggestions for both my research and my PhD study. The administrative and coordination work done by Ms. Connie Pollard, Ms. Martha Bishop and Dr. Melanie Schori was fantastic. Also, the previous and current members of the Showalter lab and Faik lab, Dr. Harjinder Sardar, Ms. Mohor Chatterjee, Mr. Nan Jiang, Mr. Brian Keppler, Mr. Richard Wiemel and Ms. Rebekah Whitley were such supportive and nice people to be around. Without the help and kindness from all the above people, my research would not have gone as smoothly and my study at OU would not have been so enjoyable. I thank the Department of Environmental and Plant Biology and the Molecular and Cellular Program for providing teaching assistant scholarships and funding for me to attend national meetings. Last but not least, I am indebted to my family and friends, who walked me through difficult times and provide me with endless love and support. 7 TABLE OF CONTENTS Page Abstract ………………………………………………………………………………….3 Acknowledgments............................................................................................................... 5 List of Tables ……………………………………………………………………………14 List of Figures ……………………………………………………………………………16 List of Abbreviations ........................................................................................................ 20 Chapter 1: Introduction ..................................................................................................... 23 1.1. Plant Cell Walls ................................................................................................. 23 1.1.1. Biological and economic importance ..................................................... 23 1.1.2. Structural model and compositions ........................................................ 24 1.1.3. Biosynthesis of cell wall polysaccharides .............................................. 31 1.2. Hydroxyproline Rich-Glycoproteins (HRGPs) .................................................. 42 1.2.1. Classification .......................................................................................... 42 1.2.2. Three major HRGP subfamilies: PRPs, EXTs and AGPs ...................... 44 1.2.3. HRGP Glycosylation .............................................................................. 47 1.3. Arabinogalactan-Proteins (AGPs) ..................................................................... 52 1.3.1. Classification .......................................................................................... 52 8 1.3.2. Post-translational modifications ............................................................. 53 1.3.3. Biological functions ............................................................................... 60 1.4. Specific Aims of the Research ........................................................................... 62 Chapter 2: Identification and Characterization of in vitro Galactosyltransferase Activities Involved in Arabinogalactan-Protein Glycosylation in Tobacco and Arabidopsis ..................................................................................................... 64 2.1. Introduction ........................................................................................................ 64 2.2. Material and Methods ........................................................................................ 68 2.2.1. Suspension culture of Arabidopsis cells ................................................. 68 2.2.2. Preparation of microsomal membranes from tobacco and Arabiodopsis suspension cultured cells........................................................................ 68 2.2.3. Standard assay for galactosyltransferase (GalT) activities ..................... 69 2.2.4. Analysis of the GalT assay products by reverse phase-high performance liquid chromatography (RP-HPLC) ......................................................