Zuzanna Dutkiewicz1, Annabelle Varrot2, Shi Yan1, Jorick Vanbeselaere1, Katharina Paschinger1, Iain B.H. Wilson1
1Department für Chemie, Universität für Bodenkultur, 1190 Wien, Austria
2Univ. Grenoble Alpes, CNRS, CERMAV, UPR5301, 601 Rue de la Chimie, 38000 Grenoble, France
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 – for none of them has a crystal structure been reported. In contrast, bioinformatics analyses have shown that only HEX-1/ HEX-3 are present in the genome of Trichuris suis (KFD57778/KFD51498). Phylogenic reconstruction indicates that these hexosaminidases evolved within the nematode branch. All nematode hexosaminidases share a common ancestor, and within the class there has been gene duplication and specialization. Homology-based 3D-structure prediction of hexosaminidases from C. elegans and T. suis showed no significant differences in the substrate binding pocket. Recent, we acquired a crystal of HEX-3 T. suis and could get diffraction at a resolution of 2.5 Å. However, this does not solve the question of the enzymes’ specificity. In vitro enzyme tests of HEX-3 T. suis showed that the enzyme favors pNP-β-GalNAc, but still can remove GlcNAc from the N-glycan substrate GnGn-PA. Thus, this Trichenelid HEX-3 enzyme is functionally more similar to HEX-2 and HEX-3 from C. elegans than to HEX-4. The latter can remove terminal GalNAc even from structures in which the underlying GlcNAc was substituted with phosphorylcholine. Overall, these nematode enzymes are not only interesting due to their roles in glycan biosynthesis, but are useful as a complement to mass-based glycomic workflows.
Keywords: hexosamnidases; glycans; enzymes; crystallography; bioinformatics