Authors

Discussion

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosidases responsible for maintaining normal cellular homeostasis by preventing the accumulation of glycan substrates on proteins and lipids.  Mutations in the genes encoding both enzymes lead to neurological and neuromuscular impairments seen in both Fabry disease and Schindler/Kanzaki disease. This current study investigates whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains enzymatically active α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni’s α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of platyhelminth α-GAL/α-NAGAL protein types showed that one particular S. mansoni protein (Smp_089290) to be quite distinct from other putative α-GAL/α-NAGAL members and the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. By homology modelling, the predicted three-dimensional structure of Smp_089290 exhibited folds and secondary features similar to those described for human α-GAL/α-NAGAL proteins. Both α-GAL and α-NAGAL enzymatic activities in adult S. mansoni worms were significantly higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with Smp_089290’s female biased gene expression. While siRNA-mediated knockdown of Smp_089290 in adult male worms caused a slight decrease in α-GAL activity, α-NAGAL activity was significantly reduced in these same samples when compared to control male worms (siLuc treated males; p<0.01). Similarly, a significant reduction in α-NAGAL activity was also exhibited in adult female worms treated with the same Smp_089290-specific siRNA when compared to siLuc treated females (p<0.05). Based on these enzymatic and functional genomics results, Smp_089290 acts predominantly as a α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites. Whole-plate motion-based screening of siSmnagal treated males and females revealed a significant decrease in motility when compared to siLuc treated controls (p<0.05). Inhibition of SmNAGAL activity could lead to glycan substrate accumulation which cause adult schistosomes to experience severe neuromuscular/motility defects consistent with the phenotypes observed in Fabry and Schindler/Kanzaki diseases. Interestingly, whole mount in situ hybridization (WISH) analysis of adult male schistosomes revealed Smnagal staining throughout the tegmental cells lining the esophagus as well as within neurons (including the cephalic ganglian). Further investigations quantifying glycan accumulation of siSmnagal treated worms (males and females) will increase our understanding of this schistosome gene product in normal developmental processes and reveal phenotypes useful for drug development efforts.