Lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells

Abstract

Sandhoff and Tay-Sachs disease are autosomal recessive GM2 gangliosidoses where a deficiency of lysosomal beta-hexosaminidase results in storage of glycoconjugates. Imino sugar (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) inhibition of beta-hexosaminidase in murine RAW264.7 macrophage-like cells led to lysosomal storage of glycoconjugates that were characterised structurally using fluorescence labelling of the free or glycolipid-derived oligosaccharides followed by HPLC and mass spectrometry. Stored glycoconjugates were confirmed as containing non-reducing GlcNAc or GalNAc residues resulting from the incomplete degradation of N-linked glycoprotein oligosaccharide and glycolipids, respectively. When substrate reduction therapeutics N-butyl-deoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) were applied to the storage phenotype cells, an increase in glucosylated and galactosylated oligosaccharide species was observed due to endoplasmic reticulum alpha-glucosidases and lysosomal beta-galactosidase inhibition, respectively. Hexosaminidase inhibition triggered a tightly regulated cytokine-mediated inflammatory response that was normalised using imino sugars NB-DNJ and NB-DGJ, which restored the GM2 ganglioside storage burden but failed to reduce the levels of GA2 glycolipid or glycoprotein-derived N-linked oligosaccharides. Using a chemically induced gangliosidosis phenotype that can be modulated with substrate lowering drugs, the critical role of GM2 ganglioside in the progression of inflammatory disease is also demonstrated.

Scalable Syntheses of Both Enantiomers of DNJNAc and DGJNAc from Glucuronolactone: The Effect of N‐Alkylation on Hexosaminidase Inhibition

“…Free oligosaccharide (FOS) analysis26 was performed on HL60 cells after treatment for 24 h with the N ‐alkyl derivatives of DGJNAc ( 1 Da , 1 Db , 1 Dc , and 1 De ), which gave additional free glycans (FOS) in the HPLC profiles that were derived from HL60 homogenates (Figure 2 b) in comparison to the untreated control reactions (Figure 2 a). Subsequent treatment of these FOSs with jack bean β‐GlcNAcase identified the newly produced glycans as non‐reducing terminal GlcNAc‐containing species; their identity was verified by comparison with the retention times in glucose units (GUs) reported by Boomkamp et al.…”

Synthesis of the enantiomers of XYLNAc and LYXNAc: comparison of β-N-acetylhexosaminidase inhibition by the 8 stereoisomers of 2-N-acetylamino-1,2,4-trideoxy-1,4-iminopentitols

“…59 Besides α-GalNAcase and HexNAcases, compounds 20-23 were also screened against an extended set of glycosidase enzymes (see the ESI †): α-glucosidase (yeast), β-glucosidase (almond), α-galactosidase (coffee beans), β-galactosidase (bovine liver), α-mannosidase (Jack bean), β-mannosidase (snail) and α-L-rhamnosidase (P. decumbens). As none of the compounds showed inhibition against any of the enzymes in this extended panel the HexNAcase inhibitors described in this paper are shown to be highly selective.…”

Biochemical and structural study on a S529V mutant acid α-glucosidase responsive to pharmacological chaperones

“…13,14,28 However, these imino sugars are potent inhibitors of a-glucosidases including GAA and intestinal maltase-glucoamylase, and the administration of an excess dose results in intracellular storage of glycogen, 29 and glucosylated and galactosylated oligosaccharides. 30 So, it seems to be difficult to determine the proper doses of these chemicals for pharmacological chaperone therapy, and inadequate doses of them would worsen Pompe disease and might lead to diarrhea. Furthermore, the mutant GAA species for which imino sugars are effective are limited.…”