The Wild team is an ATIP-funded Junior Group newly established in January 2021. Hosted by the Institut de Biologie Structurale in Grenoble, our team is part of the Structure and Activity of Glycosaminoglycan Group (SAGAG).
The team has a keen interest in understanding glycosyltransferases and glycan-modifying enzymes on a molecular level. A specific focus lies on the enzymes involved in the biosynthesis of heparan sulfate, a complex polysaccharide with diverse biological functions. We aim to dissect the architecture and catalytic mechanism of these enzymes by combining structural biology approaches, in particular single-particle cryo-electron microscopy, with in vitro functional and biophysical assays and in cellulo studies.
Heparan Sulfate Biosynthesis
Heparan sulfates are linear, but highly complex polysaccharide chains found on the cell surface of all animal cells. The polysaccharide chain is covalently linked via a serine residue to the core-protein and mediates the interaction with other cellular factors. In doing so, heparan sulfates play a role in a vast number of biological processes, including cell development, lipid metabolism, tissue repair, inflammation, immune responses and host-pathogen interaction. Malfunctioning of heparan sulfate biosynthesis has been linked to Alzheimer’s disease, acute and chronic inflammation, tumorigenesis and diabetes.
Heparan sulfate biosynthesis takes place in the Golgi lumen and involves the fine-tuned interplay of more than a dozen of membrane-anchored glycosyltransferases and glycan-modifying enzymes. Several studies showed that the sequence of heparan sulfate is cell and tissue specific and that it can change during embryonic development, diseases and aging. The regulatory mechanisms, however, remain elusive. Several of the heparan sulfate biosynthesis enzymes were shown to interact with each other and it was proposed that they might assemble into a large super complex, the so-called ‘GAGosome’ and that its composition might define the generated polysaccharide sequence.
Overview on heparan sulfate biosynthesis. A) Schematic representation of a heparan sulfate proteoglycan with enzymes involved in chain polymerization and modification indicated alongside. B) The GAGosome model describes a hypothetical complex formation of the enzymes involved in heparan sulfate biosynthesis.
Cryo-electron microscopy provides molecular insight into the mechanism of glycosyltransferases
Single-particle cryo-EM is the state of the art method to determine the structure of membrane proteins and large complexes. Recently, we solved the structure of the human EXT1-EXT2 complex that carries out a key step in heparan sulfate biosynthesis - the polymerization of the long glycan backbone. In a previous study, we determined the structure of the yeast oligosaccharyltransferase, a central enzyme complex in protein N-linked glycosylation that catalyses the transfer of a per-assembled glycan tree from its lipid linker to protein substrates.
Cryo-electron microscopy structure determination of the human heparan sulfate polymerase complex and the yeast oligosaccharyltransferase complex. A) Exemplary cryo-EM micrograph and B) selected 2D classes. C) 3D reconstruction and structure of the EXT1-EXT2 complex. Source : Leisico et al., Nature communications 2022. D) High-resolution cryo-EM structure of the yeast oligosaccharyltransferase complex. Source : Wild et al., Science 2018.
The host institute has an excellent infrastructure for pursuing challenging structural biology projects, including an EM platform that offers direct access to a Glacios microscope equipped with a K2 camera and a Tecnai F20 screening microscope. In addition, we have access to the Titan Krios microscope at the ESRF, which is located on the same campus.
Candidates for an internship, PhD or a postdoc in the team should write an email to Rebekka Wild.
Team members :
Current team members from left to right : Poushalee Dutta (PhD student), Marie Bourgeais (research technician), Rebekka Wild (team leader), Francisco Leisico (postdoctoral fellow)
Alumni team members :
Margot Weber (Master student, M1)
Farah Fouladkar (Bachelor student)
Juneina Omeiri (Master student, M2)
Borys Pedenko (Master student, M1)
Recent publications :
- Leisico F*, Omeiri J*, Le Narvor C, Beaudouin J, Hons M, Fenel D, Schoehn G, Couté Y, Bonnaffé D, Sadir R, Lortat-Jacob H#, Wild R# (2022). Structure of the human heparan sulfate polymerase complex EXT1-EXT2. Nature Communications doi : 10.1038/s41467-022-34882-6.
- Neuhaus JD*, Wild R*,#, Eyring J, Irobalieva RN, Kowal J, Lin C-W, Locher KP, Aebi M# (2021). Functional analysis of Ost3p and Ost6p containing yeast oligosaccharyltransferases. Glycobiology doi : 10.1093/glycob/cwab084.
- Ried MK*, Wild R*, Zhu J, Pipercevic J, Sturm K, Broger L, Harmel RK, Abriata LA, Hothorn LA, Fiedler D, Hiller S, Hothorn M (2021). Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis. Nature communications doi : 10.1038/s41467-020-20681-4
- Annaval T, Wild R, Crétinon Y, Sadir R, Vivès RR, Lortat-Jacob H (2020). Heparan Sulfate Proteoglycans Biosynthesis and Post Synthesis Mechanisms Combine Few Enzymes and Few Core Proteins to Generate Extensive Structural and Functional Diversity. Molecules doi : 10.3390/molecules25184215
- Wild R*, Kowal J*, Eyring J*, Ngwa EM, Aebi M, Locher KP (2018). Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation. Science doi : 10.1126/science.aar5140
- Wild R*, Gerasimaite R*, Jung JY*, Truffault V, Pavlovic I, Schmidt A, Saiardi A, Jessen HJ, Proirier Y, Hothorn M, Mayer A (2016). Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains. Science doi : 10.1126/science.aad9858
- Wild R, Hothorn M (2016). The macro domain as fusion tag for carrier-driven crystallization. Protein Science doi:10.1002/pro.3073