Nematode hexosaminidases – a new biochemical tool for glycan analysis

Hexosaminidases are ubiquitous enzymes with multiple roles in glycoconjugate metabolism as they remove non‐reducing terminal β ‐N‐acetylhexosamine residues from glycans, glycolipids, glycoproteins and glycosaminoglycans. Amongst many functions in non‐invertebrates, hexosaminidases are involved in maturation of N‐glycans. Previous studies of Caenorhabditis elegans Hexosaminidases (Gutternigg, 2007; Dragosits, 2014) prove that this organism possesses five GH20 family genes: four belong to the subfamily 1: HEX‐2; ‐3; ‐4 and ‐5, one to subfamily 2: HEX‐1. C. elegans HEX‐4 was recently shown to remove terminal GalNAc even from structures in which the underlying GlcNAc was substituted with phosphorylcholine. Now, we have begun to characterize hexosaminidase orthologues from pig parasitic nematodes, specifically Oesophagostomum dentatum , Trichuris suis and Trichinella spiralis . Bioinformatics analyses indicate that T. suis and T. spiralis have only two GH20 genes, one orthologous to HEX‐1 and one paralogous to the HEX‐2‐5, whereas the genome of O. dentatum appears to contain one orthologue each of HEX‐1‐5. Phylogenetic analyses are, however, complicated by incorrect database annotations in most nematode species. In terms of in vitro characterisation, the subfamily 1 GH20 enzyme from T. suis was expressed in yeast and observed to favor pNP‐β‐GalNAc with a pH optimum of 6.5; however, it can remove a single GlcNAc from the a1,3‐mannose of the N‐glycan substrate GnGn‐PA, as well as both GalNAc residues from a biantennary LacdiNAc‐containing structure. Thus, this enzyme appears to have the properties akin to both HEX‐2 and HEX‐4 from C. elegans . Homology‐based 3D‐structure prediction of hexosaminidases from C. elegans and T. suis showed no significant differences in the substrate binding pocket. In summary, nematode hexosaminidases have distinct substrate specificities and different numbers of homologues per species exist. These nematodes enzymes are not only interesting due to their roles in glycan biosynthesis; due to their specificities, they are useful as a complement to mass‐based glycomic workflows.