Multimolecular Glycosyltransferases complexes involved in glycosaminoglycan-Linker assembly – GlycoLink

Glycosaminoglycans (GAGs) are essential biopolymers attached to core-proteins of proteoglycans (PGs), that perform multiple structural and regulatory functions in cell and tissues of multicellular organisms. They are synthesized by the coordinated action of glycosyltransferases (GTs) and partners, which pathogenic variants lead to life-threatening genetic diseases. This process is initiated by the synthesis of a tetrasaccharide sequence that primes GAG chains polymerization. A recent paradigm put forwards the role of a multimolecular complex temptingly called “GAGosome” in ensuring fidelity and efficiency of GAG synthesis. This mechanism has been established for GAG chains polymerization and probably regulates GAG initiation, but less is known about the protein-protein interactions (PPI) between GTs (and partners) that assemble the linker region of GAGs.
GlycoLink aims to explore the formation and organization of multimolecular complexes between GTs and partners driving the assembly of the GAG-linker, and their functional impact in rare genetic diseases. We will explore the biophysical PPI between GTs (and partners) primarily those between two functionally relevant pairs of enzymes, ß3GalT6-Fam20B kinase and ß4GalT7-GlcAT-1 and assess the influence of pathogenic variants of these enzymes which cause severe osteochondrodysplasia called ‘linkeropathies’. The occurrence of homo- and heterodimers will be investigated in cellulo by fluorescence-lifetime imaging microscopy coupled with immunoprecipitation (IP) and crosslink (XL) mass spectrometry (MS) analyses of the complexes. Advanced structural bio-informatics using deep-learning methods combined with in vitro biophysical investigations based on mass photometry (MP) and surface plasmon resonance (SPR) will inform on PPI at the molecular level. In vitro GT enzyme activities using specific synthetic saccharide substrates and in cellulo GAG analyses will explore the functional aspects of PPI. An ultimate goal will be to elucidate the structure of individual enzymes in complex with their substrates and multimolecular complexes by crystallography and cryo-electron-microscopy (cryo-EM). Altogether, we expect to answer three questions: (Q1) How do GAG-linker enzymes interact in the Golgi apparatus? (Q2) What is the structural basis for such multimolecular organization? And (Q3) could its functional relevance explain the puzzling phenotype diversity observed in the ‘linkeropathies’?
We expect this pluri-disciplinary project combining biochemistry, chemistry, bio-informatics and structural biology to have a significant impact. From a fundamental perspective, GlycoLink will provide information on the nature and structure of the complexes governing the initiation of GAG chains and help decipher how the concerted action of GAG-biosynthetic enzymes, within Golgi apparatus ‘encode’ highly specific saccharide sequences. GlycoLink is also anticipated to have a significant clinical impact, providing clues for comprehending and correcting severe genetic conditions and improving patients’ care. This ambitious project will benefit from significant expertise and tools developed by our teams, which together with cutting-edge approaches in bioinformatics and structural biology brought by outstanding young researchers and the collaborations established between all partners, will ensure its success.