The plant oligosaccharyltransferase complex

In all eukaryotic cells, N-glycosylation is an essential and very common modification of proteins entering the secretory pathway. N-glycosylation affects many properties of proteins including their conformation and interaction with other proteins. This highly conserved process is characterized by the transfer of a preassembled oligosaccharide from a lipid carrier to selected asparagine residues present in a conserved amino acid sequence motif within newly synthesized proteins. A single membrane-bound multiprotein complex - called the oligosaccharyltransferase (OST) complex - catalyses the transfer of the oligosaccharide to newly synthesized protein. In yeast and metazoans, the OST complex is well studied and consists of one catalytically active subunit and several different non- catalytic subunits with accessory function. By contrast, little is known about the composition of the plant OST complex and the functional roles of many subunits remain elusive. Importantly, we have recently shown that human glycoproteins are incompletely N-glycosylated in plants. A comparison of structural features and amino acid sequence motifs from already known plant and mammalian OST subunits indicate clear differences that may explain the observed variation in N-glycosylation efficiency. Hence, it is hypothesized that the composition of the OST complex and the molecular function of individual subunits differ substantially between plants and mammals. In this project, we will investigate the N-glycosylation defect of OST mutants using a glycoproteomics approach and identify the plant OST complex composition by affinity purification coupled to mass spectrometry- based identification of proteins. Newly identified OST subunits will be explored for their involvement in N-glycosylation of proteins using biochemical methods, advanced bioimaging and phenotypic characterization of mutant plant lines. The systematic characterization of the plant OST complex will lead to a better understanding of N-glycosylation in plants and reveal plant-specific features of the complex that are currently unknown and highly relevant for biotechnological applications in the future.

The plant oligosaccharyltransferase complex In all eukaryotic cells, N-glycosylation is an essential and very common modification of proteins entering the secretory pathway. N- glycosylation affects many properties of proteins including their structure and interaction with other proteins. This highly conserved protein modification is characterized by the transfer of a preassembled oligosaccharide from a lipid carrier to selected asparagine residues present in a conserved amino acid sequence motif within newly synthesized proteins. A membrane- bound multiprotein complex - called the oligosaccharyltransferase (OST) complex - catalyses the transfer of the oligosaccharide to newly synthesized proteins. In yeast and metazoans, the OST complex is well studied and consists of one catalytically active subunit and several different non-catalytic subunits with accessory function. By contrast, little is known about the composition of the plant OST complex and the biological function of different subunits. Here, we investigated the N-glycosylation defect of different OST-deficient mutants using a glycoproteomics approach and characterized the OST complex composition in the model plant Arabidopsis thaliana by affinity purification coupled to mass spectrometry-based identification of proteins. We could show the involvement of a previously uncharacterized plant protein in N- glycosylation and identified its interaction partners. Furthermore, by mapping of specific protein-protein interactions we found novel OST subcomplexes that appear different from the mammalian OST complex. The characterisation of the plant OST complex enabled us to develop molecular tools to specifically modulate the N-glycosylation efficiency of proteins. This is of great interest for future biotechnological applications because plants are used by industry for the production of recombinant glycoproteins for therapeutic or prophylactic use.