Plant N-acetylglucosaminidases

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. In contrast to animals, plants contain a large amount of truncated N-linked oligosaccharides (paucimannosidic N-glycans), which completely lack terminal N- acetylglucosamine residues. Up to now it has not been elucidated how these structures are generated. In particular, no information is yet available on the enzyme(s) involved in this process. The aim of this project is to characterise a family of three Arabidopsis thaliana proteins, which display a significant homology to a N-glycan processing ß-N-acetylglucosaminidase present in animals. Preliminary results in our laboratory suggest that at least one of these enzymes represents a processing ß-N-acetylglucosaminidase and is involved in the formation of paucimannosidic N-glycan structures. To investigate the actual function of the protein family, the three plant genes will be cloned, 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 three enzymes in the processing of N-glycans in vivo. In this respect the N-glycan profiles will be determined in knockout lines. Furthermore subcellular localisation studies of the proteins will provide important information about their mode of action and the processing of paucimannosidic N-glycans. The obtained results will help to understand the biosynthesis and role of late N-glycan processing steps in plants.

Most plant glycoproteins contain substantial amounts of truncated N-glycans instead of their direct biosynthetic precursors, complex N-glycans with terminal N-acetylglucosamine residues. In this project we have demonstrated that two specific enzymes (ß-N-acetylhexosaminidases - HEXO1 and HEXO3) residing in different subcellular compartments jointly account for the formation of truncated N-glycans in the model plant species Arabidopsis thaliana. Total N-glycan analysis of corresponding gene-knockout plants revealed that the two characterised enzymes contribute equally to the production of the truncated N-glycans in roots, while N-glycan processing in leaves depends more heavily on HEXO3 than on HEXO1. Importantly, hexo1 hexo3 double mutants do not display any obvious phenotype under normal growth conditions as well as upon exposure to different forms of abiotic or biotic stress. Our results thus contribute significantly to the understanding of the biosynthesis and function of plant N-glycans and establish the feasibility of improving the production of glycoprotein therapeutics in plants by downregulation of endogenous ß-N-acetylhexosaminidase activities.