A. thaliana galactosyltransferase family

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. Whereas ER and early Golgi processing steps of N-glycans are highly conserved between phyla, the terminal steps, which lead to the formation of complex N- glycans differ considerably between mammals and plants. Plant complex N-linked glycans are characterised by the presence of terminal GlcNAc, beta-1,2-linked xylose and core alpha-1,3-linked fucose residues and the absence of beta-1,4-galactose and sialic acid. The only known chain elongation is the attachment of beta-1,3-galactose and alpha-1,4-fucose residues, which form a trisaccharide known as the Lewis a (Le a) epitope. Up to now it has not been elucidated how this structure is generated. In particular, no information is yet available on the enzyme(s), which catalyse(s) the transfer of galactose in beta-1,3- linkage to terminal GlcNAc residues. The aim of the project is to identify and characterise the first plant Lewis-type beta-1,3-galactosyltransferase, which initiates the formation of the Lea epitope. The A. thaliana genome contains a family of six putative galactosyltransferases, which display significant homology to beta-1,3-galactosyltransferases present in mammals. An expression cloning strategy will be used to generate the epitope in A. thaliana leaves, which do not display any Lea epitope in wildtype plants. In addition to that a reverse genetics approach will be carried out to identify putative redundant functions between the members of the gene family. The enzymes will be heterologously expressed in insect cells and a comprehensive study of the substrate specificity and linkage analyses will be performed to determine the enzymatic properties in vitro. Furthermore subcellular localisation studies of the proteins will provide important information about their mode of action and the processing of complex N-glycans. The obtained results will lead to further understanding of the biosynthesis pathway leading to the formation of complex N-glycan in plants.

N-glycosylation is one of the major posttranslational modifications of proteins in all 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. Whereas ER and early Golgi processing steps of N-glycans are highly conserved between phyla, the terminal steps, which lead to the formation of complex N-glycans differ considerably between mammals and plants. The only known chain elongation in plants is the attachment of beta-1,3-galactose and alpha-1,4-fucose residues, which form a trisaccharide known as the Lewis a epitope. In this project we report the identification of an Arabidopsis thaliana beta1,3- galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single protein (named GALACTOSYLTRANSFERASE1) that increased the originally very low Lewis a epitope levels in planta. Characterization of the enzymatic properties as well as subcellular localization studies showed that the identified protein is capable to transfer galactose residues to various N-glycan substrates and resides in the trans-Golgi apparatus of plant cells. Our results contribute to the understanding of the biosynthesis and function of complex N- glycan modifications, which is important for basic research and the production of biopharmaceuticals in plants.