Glucosylceramide metabolism: from 3D structure to the development of selective chemical probes

Gaucher's disease, the most prevalent of the lysosomal storage disorders, is caused by insufficient lysosomal glucocerebrosidase (GBA1) activity. This is the result of point mutations in the encoding gene, GBA1. The consequence of this reduction in activity is an accumulation of GBA1's substrate, glucosylceramide, in the lysosomes, leading to the various pathologies of Gaucher’s disease. Treatment approaches for Gaucher's disease range include enzyme replacement therapy, substrate reduction therapy and the use of small molecules to stabilise mutant forms of the enzyme – pharmacological chaperone therapy. Diagnosis and treatment of Gaucher’s disease requires regular quantification of the active GBA1 in a patient's tissues, not just the total GBA1 concentration or total β-glucosidase activity. Human cells also contain a secondary, non-lysosomal glucocerebrosidase, GBA2. The activity of GBA2 can affect the pathology of Gaucher's disease, and GBA2 may interact with some chaperones and probes targeted for GBA1. Point mutations occurring in GBA2 are also linked to human diseases including hereditary spastic paraplegia and cerebellar ataxia. In this thesis, I describe the three-dimensional structures of human GBA1 and a bacterial homologue for GBA2, TxGH116 – both unliganded and bound to a variety of probe and inhibitor compounds. Prior to this work the only structure of GBA1 with a covalently bound inhibitor were complexes with conduritol-β-epoxide. Here I describe GBA1 in complex with both gluco- and galacto-configured aziridines. The reporting of the structure of TxGH116, which was the first GH116 family protein to be structurally characterised, lead to the founding of CAZy structural clan GH-O. The structural knowledge of these two glucocerebrosidase proteins, especially in complex with covalent inhibitors, can be used to aid the design of probes and eventually drugs with improved specificity for GBA1 or GBA2.