Mapping the epitope of a selected nanobody 2BAV95 on the heterodimeric complex EXT1-EXT2 for future applications in cellulo

We aim at labeling, visualizing and studying in vivo, in the Golgi of mammalian cells, glycosyltransferases responsible for the biosynthesis of glycosaminoglycans (GAGs), long polysaccharides involved in a plethora of key cellular mechanisms. In particular, the enzymatic complex EXT1-EXT2 polymerises the GAG heparan sulfate (HS). Deficiencies in HS levels in cells has been correlated to severe diseases, including breast cancer development.
Although EXT1-EXT2 has been investigated in vitro, many questions remain opened: EXT1-EXT2 regulation in the cell, EXT1-EXT2 interaction with enzymes involved in HS chain modification, formation of supra-molecular assemblies in the Golgi.
Our labeling strategy involves nanobodies specific to EXT1-EXT2. We seek first to characterize complexes formed between selected nanobodies and EXT1-EXT2 by SPA. Indeed, we need to validate that nanobody binding does not impact EXT1-EXT2 proper localization in the Golgi, or do not block the active sites of EXT1-EXT2