Hans Bakker is a biochemist and established glycobiologist with expertise in glycosyltransferases and is especially interested in the the specific protein glycosylation of small repeats. Over the last years, he has identified several new glycosyltransferases including the xylosyltransferases responsible for glycosylation of Notch EGF repeats (Sethi et al., 2012; Sethi et al., 2010) and the C-mannosyltransferase (Buettner et al., 2013). After cloning the first C-ManT (Buettner et al., 2013). Whereas C. elegans has one C-ManT (DPY-19), four homologs are present in mammals. The Bakker group meanwhile showed that two of the mammalian homologs (DPY19L1 and DPY19L3) modify different tryptophans in the tryptophan-arginine ladder of thrombospondin repeats and are, therefore, both required for full mannosylation of the WxxWxxWxxC motif (Shcherbakova et al 2017).

 

Britta Brügger is a specialist in membrane biochemistry and was among the pioneers implementing the novel field of lipidomics (Brugger, 2014; Brugger et al., 1997). Her group has longstanding expertise in biochemistry, chemical biology and cell biology, and has developed key technologies to study protein-lipid interactions and to quantify lipids by nano-mass spectrometry. Her group discovered a novel mechanism of how lipids can modulate the activity of membrane proteins (Contreras et al., 2012), and described sphingolipid binding motifs in multi-spanning membrane proteins, among them glycosyltransferases (Bjorkholm et al., 2014). Building on the hypothesis that glycosylation defects also affect lipid synthesis, the Brügger group, in collaboration with P9 Thiel, performed lipidomics analyses of different CDG models, and found that indeed lipid homeostasis is perturbed in some CDG patients.

 

Falk Büttner is a glycobiologist with a strong background in analytics who recently developed a novel approach for the analysis of glycosphingolipid glycosylation (Rossdam et al., 2019). He has contributed to the identification and characterization of several glycosyltransferases including the first C-mannosyltransferase, DPY-19 from C. elegans (Buettner et al., 2013). His research focuses on the analysis of the glycome and proteome during early human development using human pluripotent stem cells . A model for PMM2-CDG has been established in his group by reprogramming PMM2-CDG patient-derived fibroblasts into iPSCs (Thiesler et al., 2016). The PMM2-iPSC model together with the identification of the C-mannosyltransferase will enable to decipher the impact of PMM2-CDG on other types of dolichol-based glycosylation and their effects on development.

 

Erdmann Rapp is analytical chemist and renowned for the development and implementation of innovative cutting edge bio(process)analytical tools for a better understanding of bio(techno)logical processes – in particular, on high performance tools for proteomics, glycoproteomics, and glycomics to enable in-depth analysis of complex samples and digging deeper into glycobio(techno)logy. He and his team have developed, validated, implemented and are applying a comprehensive set of high-performance tools (Hoffmann et al., 2016; Hennig et al., 2016; Thiesler et al., 2016; Pralow et al., 2017; Nguyen-Khuong et al., 2018; Hoffmann et al., 2018; Reiding et al., 2019). This toolbox, consisting of orthogonal (electrophoresis-, chromatography- and/or mass spectrometry-based) methods and dedicated software-tools (Pioch et al., 2018), enables them to study all types of glycosylation in the context of this proposal.

 

Thomas Ruppert has longstanding expertise in mass spectrometric protein characterization (Iyer-Bierhoff et al., 2018; Sobotta et al., 2015; Uddin et al., 2019) and quantitative proteomics (Bärenz et al., 2013; Wei et al., 2019). Three different workflows for reliable data interpretation of discovery based and targeted proteomics strategies (Proteome Discoverer workflow, MaxQuant/Perseus and Skyline) are established to meet the requirements of different biological questions. In collaboration with the Strahl group, his lab recently developed a new method for the discovery of mammalian O-mannose glycopeptides. To overcome the limitations of mass spectrometry in differentiating glycan isoforms, the specificity of a mannosyl glycosidase was used in combination with quantitative mass spectrometry to prove that hexosylated peptides are mannosylated .

 

Harald Schwalbe is a biological chemist and investigates the structure and dynamics of biomolecules (protein and RNA), in particular by nuclear magnetic resonance spectroscopy but also other biophysical techniques. Work is conducted at the Center for Biomolecular Magnetic Resonance (BMRZ), a European research infrastructure with sixteen NMR spectrometers from 600-950 MHz equipped with cryogenic probes. The group has pioneered methods to express isotope labeled proteins in insect cells, Pichia. pastoris and HEK293 cells, (Dutta et al., 2012; Klein-Seetharaman et al., 2004; Saxena et al., 2012; Stehle et al., 2012; Werner et al., 2008) allowing the rapid measurement of that promising NMR spectra of isotopically labelled C-mannosylated and non-mannosylated proteins (unpublished data). His group is interested in understanding the influence of glycosylation on the structure, function and dynamics of proteins by NMR in solution.

 

Irmgard Sinning is an expert in integrative structural biology with a longstanding interest in membrane protein biogenesis and protein transport (Stefer et al. 2011; Grotwinkel et al. 2014; Jadhav et al. 2015; Becker et al. 2017; Wild et al. 2019). She has been centrally involved in establishing a cryo-EM Network at Heidelberg University to meet the increasing need for cryo-electron microscopy, and attracted about 4 Mio € for instrumentation. Her group uses X-ray crystallography and now also cryo-EM together with biochemical and biophysical methods to dissect the structure and dynamics of molecular machines. They run a crystallization platform and are experts in membrane protein expression (e.g. Hackmann et al., 2015). Starting from the structure of the SDF2 protein (Schott et al., 2010) they characterized different Pmt MIR domains and embarked on an in depth analysis of mannosyltransferases.

 

Sabine Strahl is a glycobiologist who´s laboratory has substantially contributed to the basic understanding of protein O-Man in yeast and mammals. Besides comprehensive cell biological and biochemical know-how for glyco- and membrane proteins, the lab has established a variety of “glyco-specific” tools. Work from her lab qualified yeast Pmt4 as an ideal model to study mammalian O-mannosyltransferases (Bausewein et al., 2016). The group recently unraveled the first yeast O-mannose glycoproteome and showed that O-mannosyl glycans affect far more processes than previously thought (Neubert et al., 2016; Zatorska et al., 2017; Castells Ballester et al., 2018; Castells Ballester et al., 2019). The lab identified new O-Man clients from mouse brain and showed that POMTs affect human mesenchymal stem cell fate (Ragni et al., 2016) and impact cadherin-mediated cell adhesion (Lommel et al., 2013; Carvalho et al., 2016).

 

Christian Thiel is a biochemist who has been working in the field of N-Glyco deficiencies for over 20 years. He has identified several new types of CDGs including ALG1-CDG (Schwarz et al., 2004), ALG2-CDG (Thiel et al., 2003) and ALG11-CDG (Rind et al., 2010; Thiel et al., 2012) His group showed for the first time that, comparably to yeast, mammalian ALG2 and ALG11 have dual functions in transferring mannose residues. Furthermore, the Thiel group has created highly relevant CDG animal models including PMM2 knock out and hypomorphic mice and PMM2-CDG frogs, which led to the first therapeutic intervention for this type of disease in vivo (Himmelreich et al., 2015; Schneider et al., 2011; Thiel et al., 2006). Beside broad state-of-the-art techniques in biochemistry, genetics and histology, the group provides a large collection of CDG patients fibroblasts, S2- and isotope laboratories.

 

Thomas Thumberger is a developmental biologist who is interested in the molecular mechanisms driving organ formation and growth/regeneration. He has longstanding experience in using small animal model systems to investigate the molecular mechanisms leading to human disease phenotypes like ciliopathies (Inoue et al., 2015; Beyer et al., 2012;  Thumberger et al., 2012) and developmental phenotypes for instance the formation of conjoined twins (Tisler et al., 2018). He established the CRISPR/Cas technique for use in the Japanese rice fish medaka (Oryzias latipes; Stemmer et al., 2015) and refined genome editing to facilitate precise integration/modification of defined DNA donors (Gutierrez-Triana et al., 2018). In combination with advanced in vivo imaging, he addresses the molecular basis for the balance of proliferation and differentiation during growth and development (Höckendorf et al., 2012).

 

Joachim Wittbrodt is a developmental geneticist and stem cell biologist working on the molecular mechanisms governing the balance between proliferation and differentiation. He has addressed key mechanisms acting in neural development, growth and regeneration (Lust and Wittbrodt., 2018, Tsingos et al., 2019). He has long-standing experience in the use of fish model systems (Wittbrodt et al., 2002) and combines advanced in vivo imaging (Keller et al., 2008; Rembold et al., 2006) with cutting edge genetics (Centanin et al., 2014; Centanin et al., 2011) and genomic engineering via the CRISPR/CAS technology (Stemmer et al., 2015, Gutierrez-Triana et al., 2018) to facilitate precise functional analysis with subcellular resolution in the context of the living organism. His group has used transplantation and Cre/LoxP mediated recombination to study genetically different (mutant) cells in an otherwise wild type context, a technique crucial to facilitate functional analysis of genes otherwise resulting in early embryonic lethality. The Wittbrodt lab has generated fish knock-out lines for key glycosylation enzymes by enhancer trapping (Kirchmaier et al., 2013) and via CRISPR/CAS targeting and will continue to establish a wide spectrum of mutant alleles suited for biochemical and mechanistic analyses in the organismal context.