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Transglyco: Chemistry, Enzymology and Physiology of Oligosaccharyltransferase

English title Transglyco: Chemistry, Enzymology and Physiology of Oligosaccharyltransferase
Applicant Aebi Markus
Number 147632
Funding scheme Sinergia
Research institution Institut für Mikrobiologie Departement Biologie ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Molecular Biology
Start/End 01.11.2013 - 31.05.2017
Approved amount 1'355'120.00
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All Disciplines (5)

Molecular Biology
Organic Chemistry

Keywords (6)

Protein glycosylation; Saccharomyces cerevisiae; Catalysis; Trypanosoma brucei; Inhibitors; Oligosaccharyltransferase

Lay Summary (German)

N-Glykosylierung von Proteinen ist die häufigste Modifikation von Proteinen. Im Zentrum dieses für die Zelle lebenswichtigen Prozesses steht die Oligosaccharyltransferase, ein komplex aufgebautes Enzym an der Membran des Endoplasmatischen Retikulums. Die Funktionsweise dieser "molekularen Maschine" ist unverstanden, sie soll durch Nutzung von Methoden der organischen Chemie, der Biochemie und Strukturbiologie von Membranproteinen und der mikrobiellen Glykobiologie und Genetik aufgeklärt werden.
Lay summary

Die N-gebundene Glykosylierung ist die am häufigsten vorkommende Modifikation von Proteinen in eukaryotischen Zellen: über 10'000 verschiedene Stellen von Proteinen der Maus sind mit N-gebundenen Zuckern modifiziert. Die Funktion der N-gebundenen Zucker ist äusserst vielfältig und ist am besten durch die Tatsache dargestellt, dass tierische Zellen ohne den Prozess der N-Glykosylierung nicht lebensfähig sind. Das zentrale Enzym im Prozess der N-Glykosylierung von Proteinen ist die Oligosaccharyltransferase, eine komplexe molekulare Maschine an der Membran des Endoplasmatischen Retikulums.  Dieser Membranproteinkomplex katalysiert die kovalente Verknüpfung eines komplexen Zuckers mit der Asparagin-Seitenkette innerhalb der Erkennungssequenz N-X-S/T.

Über den katalytischen Mechanismus der Oligosaccharyltransferase ist wenig bekannt; die Gründe liegen in der Komplexität der involvierten Ausgangsmoleküle (Lipid-gebundene Oligosaccharide und verschiedenste Proteine als Substrate), dem quantitativen Nachweis der Reaktionsprodukte in vivo und in vitro  sowie der Schwierigkeit, Membranproteinkomplexe zu reinigen, diese mittels biochemischer Methoden zu untersuchen und letztendlich deren Struktur mit Hilfe der  Röntgenkristallographie zu bestimmen.

Um die Funktionsweise der Oligosaccharyltransferase aufzuklären arbeiten in diesem Projekt drei Forschungsgruppen mit komplementärer Expertise zusammen: Techniken der mikrobiellen Glykobiologie und Genetik (Aebi), der Biochemie und Kristallographie von Membranproteinen (Locher) sowie der synthetischen organischen Chemie und Inhibitor-Design (Reymond) werden eingesetzt, um den Reaktionsmechanismus der Oligosaccharyltransferase aufzuklären und die Funktion der verschiedenen Komponenten dieses komplexen Enzyms aufzuklären.

Direct link to Lay Summary Last update: 31.10.2013

Responsible applicant and co-applicants



Structure and mechanism of an active lipid-linked oligosaccharide flippase
Perez Camilo, Gerber Sabina, Gerber Sabina, Boilevin Jérémy, Bucher Monika, Darbre Tamis, Aebi Markus, Reymond Jean Louis, Locher Kaspar P. (2015), Structure and mechanism of an active lipid-linked oligosaccharide flippase, in Nature, 524(7566), 433-438.

Scientific events


Title Date Place
Gordon Research Conference Glycobiology 28.02.2015 Barga, Italy

Associated projects

Number Title Start Funding scheme
173709 GlycoSTART: Structure and function of eukaryotic oligosaccharyltransferase 01.06.2017 Sinergia
144083 N-linked protein glycosylation: the eukaryotic oligosaccharyltransferase 01.11.2012 Project funding
170808 Acquisition of a Talos Arctica transmission electron microscope for single particle analysis and cryo-tomography 01.05.2017 R'EQUIP
164092 Upgrade of a Titan Krios electron cryo-microscope for single particle analysis and tomography 01.12.2015 R'EQUIP
162636 N-linked protein glycosylation: generating diversity 01.11.2015 Project funding
166672 Structural and mechanistic studies of components of bacterial protein N-glycosylation pathway and of vitamin B12 transport 01.04.2016 Project funding
164025 Automated Peptide Purification System 01.01.2016 R'EQUIP


The most abundant modification of proteins in eukaryotes is N-linked glycosylation:more than 10’000 different acceptor sites are N-glycosylated in the mouse proteome. The process is essential because it underpins the folding and quality control of non-cytoplasmic (organelle-targeted), membrane-embedded, or secreted proteins. N-glycosylation is involved in organism development, the immune response, and host-pathogen interactions. Finally, a multitude of diseases are linked to the dysfunction of this process, including the various congenital disorders of glycosylation (CDGs).This proposal focuses on the central enzyme in the N-glycosylation pathway, oligosaccharyltransferase (OST). OST is a complex molecular machine that is embedded in the membrane of the Endoplasmic Reticulum (ER), where it catalyzes the transfer of a glycan moiety from a lipid-linked oligosaccharide (LLO) onto acceptor proteins that contain a recognition sequence N-X-S/T (the glycosylation sequon). After its covalent attachment to the protein, the N-linked glycan is processed both in the ER and in the Golgi compartment by glycosylhydrolases and glycosyltransferases, thereby generating the vast structural and functional diversity in eukaryotic glycoproteins. In contrast to the later events in the Golgi, which are species- or even cell type-specific, the OST-catalyzed N-glycosylation reaction in the ER is highly conserved in eukaryotes. Despite its essential role in cellular physiology, amazingly little is known about the mechanism of OST-catalyzed N-glycosylation. There are multiple reasons for this lack of knowledge: First, the physiological output of the process (the N-glycoproteome) is highly complex both in terms of the large number of modified protein substrates (including many membrane proteins) as well as the diversity in the final N-glycan. Second, OST is a dynamic, multi-subunit, integral membrane protein complex, whose over-expression and purification are highly challenging. Third, ER-based N-glycosylation is coupled to the formation of disulfide bridges and protein folding, suggesting that OST catalyzes not only one, but multiple reactions simultaneously. Fourth, the glycan donor substrate, lipid-linked oligosaccharide, is water-insoluble and of limited stability. To understand OST-catalyzed N-glycosylation, we plan to combine our expertise in (i) microbial glycobiology and genetics (Aebi), (ii) membrane protein biochemistry and crystallography (Locher), and (iii) synthetic chemistry and inhibitor design (Reymond). The trans-disciplinarity and the close collaboration between the participating groups are essential components of the projects. The very ambitious objectives of our projects can only be reached in a long-term collaborative effort. Objective 1: We plan to establish the catalytic reaction mechanism of single subunit oligosaccharyl-transferase and exploit this knowledge for engineering and inhibitor design. This includes various aspects:•Establishing the chemical mechanism of acceptor asparagine activation and glycan transfer from the LLO molecule to the glycosylation sequon.•Revealing the molecular basis of substrate recognition (peptide and LLO) using structural and biochemical methods combined with chemical synthesis.•Expanding the accepted substrates using synthetic substrate analogues as well as engineered OST variants, thus generating novel glycoproteins or glyco-conjugates for diagnostic and therapeutic purposes.•Visualizing, at high resolution, structural differences between bacterial and eukaryotic single subunit OST that account for the distinct substrate specificities. Establishing whether bacterial and eukaryotic OST indeed have the same chemical reaction mechanism.•Design of novel inhibitors that are specific for bacterial OST and have a potential application as antibiotics.Objective 2: We plan to determine the mechanism, the functional output, the structure and the subunit interactions of eukaryotic, octameric OST. This includes the following aspects:•Establishing tools for quantitatively determining the in vivo output of OST-catalyzed N-glycosylation.•Establishing the arrangement of the OST subunits, their stoichiometry and interactions in the complex, as well as the mechanism of complex assembly.•Determining the contributions of the various subunits to substrate (polypeptide and LLO) specificity and to the glycosylation reaction.•Visualizing the octameric OST structure.