F M Platt

The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding

Glucosylated oligomannose N‐linked oligosaccharides (GlcxMan9GlcNAc2 where x = 1–3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N‐glycosylation and chaperone recognition and (ii) substrates in the initial steps of N‐glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the α‐glucosidase inhibitor N‐butyl‐deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well‐defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3 Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9 GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.

Glycosphingolipid lysosomal storage diseases: therapy and pathogenesis

Paediatric neurodegenerative diseases are frequently caused by inborn errors in glycosphingolipid (GSL) catabolism and are collectively termed the glycosphingolipidoses. GSL catabolism occurs in the lysosome and a defect in an enzyme involved in GSL degradation leads to the lysosomal storage of its substrate(s). GSLs are abundantly expressed in the central nervous system (CNS) and the disorders frequently have a progressive neurodegenerative course. Our understanding of pathogenesis in these diseases is incomplete and currently few options exist for therapy.

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

Background: GBA2 and GBA are both β-glucosidases that degrade glucosylceramide. Results: Conduritol B epoxide inactivates both GBA and GBA2, whereas the imino sugar NB-DGJ selectively inhibits GBA2. Conclusion: NB-DGJ is a suitable reagent to distinguish GBA2 from GBA. Significance: This study redefines GBA2 activity, which is relevant for clinical GBA2 measurements and imino sugar pharmacology. β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher KI) and a lower rate of enzyme inactivation (kinact). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5–6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.

New Therapeutics for the Treatment of Glycosphingolipid Lysosomal Storage Diseases

Glycosphingolipid lysosomal storage diseases are a small but challenging group of human disorders to treat. Although these appear to be monogenic disorders where the catalytic activity of enzymes in glycosphingolipid catabolism is impaired, the presentation and severity of disease is heterogeneous. Treatment is often restricted to palliative care, but in some disorders enzyme replacement does offer a significant clinical improvement of disease severity. An alternative therapeutic approach termed substrate deprivation or substrate reduction therapy (SRT) aims to reduce cellular glycosphingolipid biosynthesis to match the impairment in catalytic activity seen in lysosomal storage disorders. N-Alkylated imino sugars are nitrogen containing polyhydroxylated heterocycles that have inhibitory activity against the first enzyme in the pathway for glucosylating sphingolipid in eukaryotic cells, ceramide-specific glucosyltransferase. The use of N-alkylated imino sugars to establish SRT as an alternative therapeutic strategy is described in cell culture and gene knockout mouse disease models. One imino sugar, N-butyl-DNJ (NB-DNJ) has been used in clinical trials for type 1 Gaucher disease and has shown to be an effective and safe therapy for this disorder. The results of these trials and the prospects of improvement to the design of imino sugar compounds for treating Gaucher and other glycosphingolipid lysosomal storage disorders will be discussed.