Schistosoma mansoni α-N-acetylgalactosaminidase (SmNAGAL) regulates coordinated parasite movement and egg production

Abstract

α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important 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 putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p<0.05; α-NAGAL > α-GAL), which was consistent with smp_089290’s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in α-GAL activities were observed in the same extracts. Despite this, decreases in α-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an α-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.

Transgenesis in parasitic helminths: a brief history and prospects for the future

“…Finally, Hulme et al employed both RNAi and programmed gene editing approaches in parallel to demonstrate that the glycosyl hydrolase S . mansoni α- N -acetylgalactosaminidase (SmNAGAL) plays a central role in the cellular machinery of glycan processing and modification, with dysregulation of coordinated parasite movement and egg production following both gene silencing or gene knockout of SmNAGAL [ 105 ].…”

Knockout of liver fluke granulin, Ov-grn-1, impedes malignant transformation during chronic infection with Opisthorchis viverrini

“…A third construct, termed pCas- Ov -scramble was also prepared and included as a control to normalize analysis of gene expression and programmed gene knockout. The pCas -Ov- scramble construct included as the gRNA, a transcript of 20 nt, 5’-GCACTACCAGAGCTAACTCA which exhibits only minimal identity to the O. viverrini genome and which lacks a PAM (73). A non-targeting guide RNA (26), encoded here by pCas- Ov -scramble, provided a negative control for off-targeting by the Cas9 nuclease.…”

Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR associated protein 9-mediated editing of Schistosoma mansoni genes: Identifying genes for immunologically potent drug and vaccine development

“…In humans, the lysosomal exoglycosidase enzyme, α-N-acetylgalactosaminidase (α-NAGAL), cleaves terminal N-acetylgalactosamine (α-GalNAc) monosaccharides from polysaccharides, glycoproteins, and glycolipids, including those with O-linked carbohydrate sugars attached to serine and threonine residues, and plays an important role in maintaining cellular homeostasis by regulating glycan substrates on carbohydrates, lipids, and proteins 19,67 . It belongs to the glycosyl hydrolase family 27 and contains both an α-galactosidase (melibiase)-2 (PF16499) domain and a melibiase-2 C-terminal (PF17450) domain (all enzymatically important catalytic and ligand binding amino acids are conserved within this region) 67 .…”

“…Using sequence and phylogenetic analyses, Hulme et al (2021) investigated the genome of S. mansoni to identify glycosyl hydrolase family 27 orthologs of Homo sapiens α-galactosidase and α-NAGAL enzymes 19 . Five glycosyl hydrolase family 27 members were found in S. mansoni, but only one, namely SmNAGAL (Smp_089290), contained all 13 ligand-binding amino acid residues and both catalytic aspartic acid residues that are crucial for α-galactosidase/α-NAGAL substrate cleavage and activity.…”

“…Recently, CRISPR/Cas9-mediated gene editing tools have proven to be useful in understanding the pathophysiological, metabolic, and immunological mechanisms underlying the manifestation and severity of schistosomiasis as well as in highlighting the importance of developing vaccines and drugs with high therapeutic efficacy (Table 2) (Figure 1) 4,[16][17][18][19][20] . This review aims to summarize the major findings from the current literature reporting the usage of the CRISPR/Cas9-mediated gene editing tool to inactivate genes in S. mansoni to identify the functional role of these genes in disease pathogenesis and to highlight the importance of developing vaccines and drugs with high therapeutic efficacy to target disease-associated genes.…”