N-glycosylation in plants: science and application.

In this thesis we set out to increase our knowledge of N-glycosylation in plants with a dual aim: (1) to develop tools for general and cell-specific glycoproteomics by which differences in the glycoproteome of plants in different specified cell types or under different conditions can be studied and (2) to increase our general understanding of N-glycosylation by analysis of the biosynthesis pathway and dissection of N-glycan function in plants. (1) Identification of the glycoproteome from specific cell types during different physiological or developmental conditions provides valuable biological information. For the detection of glycoproteins with plant complex type glycans anti-HRP polyclonal serum is available. Using these antibodies differences were identified in the complex glycoproteome of leaf epidermal and mesophyll cells and it was demonstrated that these differences vary in other plant tissues. Cell specific tagging with complex glycans was accomplished by cell specific complementation of the cgl mutant, demonstrating the power of this technique to detect subtle differences within a tissue that are lost in whole tissue protein extracts of wild-type plants. With anti-HRP we were not able to purify and validate the complex glycoproteome as predicted by bioinformatics. For glycoproteomics, the availability of probes that interact with specific plant N-glycans would be very helpful for the isolation and subsequent analysis of specific sub-pools of glycoproteins. In addition, such probes can aid the analysis of glycan modification on target proteins. For this reason, we used two alternative approaches to isolate monoclonal antibodies that can recognise plant complex N-glycans, neither of which yielded a probe with the desired properties. Suggestions are given on how strategies to select specific plant N-glycan antibodies may be improved. (2) N-glycan synthesis and processing is performed by sequential activity of enzymes in the ER and Golgi. Two of these enzymes were studied in more detail in this thesis: ALG3, involved in lipid-linked glycan synthesis in the ER, and GnTI involved in Golgi localized N-glycan processing to complex glycans. The ALG3 gene of Arabidopsis was identified and characterization of an alg3 mutant provided information on the in vivo substrate characteristics of various downstream enzymes, including the OST complex, and of consequences for the Unfolded Protein Response (UPR) in plants. Analysis indicated that ER resident glycoproteins from this mutant have predominantly Man3-5GlcNAc2 N-glycans. For this reason the alg3-2 mutant was used to produce an ER retained variant of monoclonal antibodies in seeds. It was demonstrated that human GnTI is much less efficient in complementing the cgl (GnTI) mutant in Arabidopsis than the homologous Arabidopsis gene. Analysis showed that this was not due to RNA expression but to differences in protein stability and reduced catalytic activity of Human GnTI. The results also suggest some form of competition between human and plant GnTI when produced in the same cell, leading to partially mutual exclusive targeting to presumed Golgi stacks.