Ceramide glucosyltransferase (CGT) is a key enzyme in glycosphingolipid (GSL)
biosynthesis in eukaryotic cells. Inhibition of enzyme activity by an N-alkylated imino sugar,
N-butyl-deoxynojirimycin (NB-DNJ), has been evaluated for the therapeutic treatment of
inherited glycosphingolipid lysosomal storage diseases. To develop more selective drugs for
potential clinical use, further investigation of possible side effects and the design of a more
selective inhibitor is required.
One concern for clinical use of NB-DNJ is the potential activation of CGT in vivo.
When rats were treated with various concentrations of NB-DNJ for 13 weeks to assess the
depletion of glycosphingolipids and up-regulation of CGT activity, the reduction of
ganglioside levels was observed following an increase in NB-DNJ dose level up to 180
mg/kg/day. However, CGT activity levels were not significantly affected by NB-DNJ treatment.
The lack of CGT up-regulation while reducing GSLs by NB-DNJ would be desirable in the
clinic to avoid a rapid accumulation of GSLs if patient treatment was concluded.
To aid in design of highly selective inhibitors for CGT, enzyme kinetic studies were
performed using recombinant human CGT and five different imino sugars. The recombinant
enzyme showed similar enzyme kinetics to a native enzyme from HL-60 cells. All the tested
imino sugars showed a mixed-type inhibition for ceramide, and an increase in N-alkyl chain
provided an improved uncompetitive inhibition. These data suggest that CGT may have two
different sites for binding of imino sugars, and the N-alkyl chain length may affect the
preference for binding site.
When the protein sequence of CGT was analysed using www server programs to predict
protein structure, a Rossman fold was predicted in the nucleotide-binding domain as observed
in other nucleotide-sugar glycosyltransferase structures. Also, a significant folding similarity to
bacterial glycosyltransferase SpsA was predicted. Based on these observations, a possible
inhibitor-binding mechanism is discussed that may aid the design of highly selective inhibitors
for CGT.</p