Mechanistic Insight into Glycosaminoglycan Chain Polymerization – from Molecules to Cells – GlycoPol

Glycosaminoglycans, such as heparan sulfate and chondroitin sulfate, are long, complex polysaccharide chains found on the surface of all animal cells. They are covalently attached to a core protein and mediate the interaction with diverse cellular factors, including chemokines, pathogens and signalling receptors. An immense diversity in functional and pathological roles is associated with the complex glycosaminoglycan composition. A key step of glycosaminoglycan biosynthesis, which takes place in the Golgi lumen, is the polysaccharide chain elongation. Its molecular mechanism, however, is still unknown. Moreover, the intrinsic feature of the core protein mediating the specific addition of either a heparan sulfate or chondroitin sulfate chain remains to be elucidated.
The objectives of this proposal are (i) to reveal the molecular basis of heparan sulfate and chondroitin sulfate chain polymerization, (ii) to uncover the intrinsic factor in the core protein that decides the fate of the generated glycosaminoglycan chain and (iii) to study the architecture of the heparan sulfate and chondroitin sulfate polymerase complexes in their native cellular environment.
To achieve these aims, in vitro glycosyltransferase assays will be combined with in cellulo functional analysis. A tetrasaccharide-peptide library will be synthesized using a chemo-enzymatic approach, and high-resolution structures of substrate-bound complexes will be determined by single-particle cryo-electron microscopy and X-ray crystallography. Golgi-localized glycosyltransferase complexes will be characterized in situ using multitask nanobodies and cryo-electron tomography.
This project will provide a comprehensive understanding of glycosaminoglycan chain polymerization across the biological scales, from molecules to cells, laying the corner stone for future research on the glycosaminoglycan function in health and disease.