Arabidopsis Mannosidase Family
Abstract
N-glycosylation is one of the major posttranslational modifications of proteins in eukaryotic cells. The biosynthesis of protein N-linked glycans results from a series of highly co-ordinated step-by-step enzymatic conversions occurring in the endoplasmic reticulum (ER) and Golgi apparatus. While the enzymes involved in the formation and processing of complex N-glycans in plants have been cloned and characterised recently, almost nothing is currently known about plant class I alpha-mannosidases, which are involved in the early N-glycan processing steps. In addition, these proteins might also play a role in N-glycan dependent quality control in the ER, where misfolded glycoproteins are destroyed by a process called endoplasmic reticulum-associated degradation (ERAD). The aim of this project is to characterise a family of five Arabidopsis thaliana proteins, which display significant homology to class I alpha-mannosidases present in mammals and yeast. Preliminary results in our laboratory suggest that these putative enzymes are involved in the trimming of Man9GlcNAc2 to Man5GlcNAc2 N-glycans. Man5GlcNAc2 is subsequently the substrate for the formation of hybrid and complex N-glycans in the Golgi. To investigate the actual function of the A. thaliana class I alpha-mannosidase family, the five plant genes will be expressed in insect cells and a comprehensive study of the substrate specificity will be carried out to determine their enzymatic properties in vitro. A reverse genetic approach will be used to investigate the possible role of the five enzymes in the processing of N-glycans in vivo. In this respect the N-glycan profiles will be determined in knockout lines. Since these proteins could also be involved in glycoprotein degradation in the ER, the expression and half-life of a well-characterised glycoprotein will be studied in the knockouts and corresponding overexpression lines. Furthermore subcellular localisation studies of the proteins and phenotypic characterisation of knockout and overexpression lines will provide important information about their putative physiological role. The obtained results will help to understand the function of early N-glycan processing steps in plants and elucidate if these class I alpha-mannosidase proteins are involved in N-glycan dependent protein degradation like it has been proposed for mammals and yeast.
Publikationen
Project staff
Richard Strasser
Assoc. Prof. Dr. Richard Strasser
richard.strasser@boku.ac.at
Tel: +43 1 47654-94345
Project Leader
01.10.2008 - 31.03.2013