Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases

Glycobiology, 2015, vol. 25, no. 4, 448–464 doi: 10.1093/glycob/cwu132 Advance Access Publication Date: 8 December 2014 Original Article Original Article Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases Martin Dragosits2,3, Shi Yan2, Ebrahim Razzazi-Fazeli3, Iain B H Wilson2, and Dubravko Rendic1,2 2 Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, and 3VetCore Facility for Research, University of Veterinary Medicine, Vienna, Austria 1 To whom correspondence should be addressed: Tel: +43-147-654-2176; Fax: +43-147-654-6076; e-mail: dubravko.rendic@ boku.ac.at Received 29 June 2014; Revised 27 November 2014; Accepted 1 December 2014 Abstract Fused lobes (FDL) hexosaminidases are the most recently genetically defined glycosidases involved in the biosynthesis of N-glycans in invertebrates, and their narrow specificity is essential for the generation of paucimannosidic N-glycans in insects. In this study, we explored the potential of FDL hexosaminidases in the utilization of different artificial and natural substrates, both as purified, native compounds or generated in vitro using various relevant glycosyltransferases. In addition to the already-known FDL enzyme from Drosophila melanogaster, we now have identified and characterized the Apis mellifera FDL homolog. The enzymatic properties of the soluble forms of the affinity-purified insect FDL enzymes, expressed in both yeast and insect cells, were compared with those of the phylogenetically distinct recombinant Caenorhabditis elegans FDL-like enzymes and the N-acetylgalactosamine (GalNAc)-specific Caenorhabditis hexosaminidase HEX-4. In tests with a range of substrates, including natural N-glycans, we show that the invertebrate FDL(-like) enzymes are highly specific for N-acetylglucosamine attached to the α1,3-mannose, but under extreme conditions also remove other terminal GalNAc and N-acetylglucosamine residues. Recombinant FDL also proved useful in the analysis of complex mixtures of N-glycans originating from wild-type and mutant Caenorhabditis strains, thereby aiding isomeric definition of paucimannosidic and hybrid N-glycans in this organism. Furthermore, differences in activity and specificity were shown for two site-directed mutants of Drosophila FDL, compatible with the high structural similarity of chitinolytic and N-glycan degrading exohexosaminidases in insects. Our studies are another indication for the variety of structural and function aspects in the GH20 hexosaminidase family important for both catabolism and biosynthesis of glycoconjugates in eukaryotes. Key words: fused lobes, hexosaminidase, insect, invertebrate, N-glycans Introduction One of the most obvious features of invertebrate N-glycomes is the presence of paucimannosidic structures, i.e. N-glycan structures consisting of just (un)modified tri- or bimannosylchitobiosyl cores. The enzymes essential for the final steps of the biosynthesis of these structures are hexosaminidases which remove the non-reducing © The Author 2014. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 448 449 Invertebrate N-glycan processing hexosaminidases the Golgi apparatus. On the other hand, even though the major, mature plant N-glycan structures resemble those found in insects [e.g. Man3GlcNAc2FucXyl N-glycan structure found in flowers of Arabidopsis thaliana and Nicotiana benthamiana (Rendic et al. 2007) in comparison with the Man3GlcNAc2Fuc N-glycan structure found in Drosophila (Fabini et al. 2001; Rendic et al. 2006)], the biosynthesis pathway of these structures in plants does not appear to involve FDL-like enzymes. Indeed, the hexosaminidases described in plants to date are predicted to be localized in the vacuole/plasma membrane (Vitale and Chrispeels 1984; Gutternigg et al. 2007; Liebminger et al. 2011) or to be involved in chitin degradation (Gutternigg et al. 2007). Furthermore, secreted plant glycoproteins (such as laccase) often contain extended structures with “Lewis a” epitopes (Fitchette-Laine et al. 1997). Insects, apart from the FDL enzymes, also express other hexosaminidases. In Drosophila, the hexosaminidases Hexo1 and Hexo2 were shown to act on chitin-derived substrates and actually are unable to remove non-reducing terminal GlcNAc residues from a typical N-glycan structure (Léonard et al. 2006). More recently, it was shown that the Hexo1 is responsible for removal of one of the two residual GlcNAc residues from the degraded N-glycan during biosynthesis of the Drosophila rhodopsin 1 (Rosenbaum et al. 2014). In contrast, both described non-FDL hexosaminidases from S. frugiperda are also able to process typical biantennary N-glycan structures (Tomiya et al. 2006; Geisler et al. 2008). In this study, we have investigated the suitability of FDL hexosaminidases in the analysis of N-glycans. For the first time, purified forms of the recombinant FDL(-like) enzymes from Caenorhabditis and Drosophila were used to measure and compare their activity toward various p-nitrophenyl-monosaccharides. Also, as an important extension of our previous work, we were able to identify and characterize the Apis mellifera homolog of the FDL hexosaminidase. Differently modified N-glycopeptides terminating with either GlcNAc or N-acetylgalactosamine (GalNAc) residues were prepared and tested as substrates for all enzymes used in this study. A comparison of the Drosophila, Caenorhabditis and Apis FDL(-like) hexosaminidases is provided. Furthermore, analysis of site-directed mutants of Drosophila FDL indicates a high structural similarity of chitin- and N-glycan degrading hexosaminidases in insects. Finally, we have clarified the position of the terminal, non-reducing β-GlcNAc on a number of N-glycans carrying this residue by utilizing the purified Drosophila FDL hexosaminidase for the structural analysis of the complex mixtures of N-glycans from wild-type and mutant Caenorhabditis strains. Results Production of recombinant FDL enzymes In an effort to produce the recombinant FDL enzyme of high purity suitable for a thorough study of its properties, we have analyzed the expression of various FDL(-like) enzymes using two different expression systems. Prompted by the success in the previous study (Léonard et al. 2006), we have initially expressed the D. melanogaster FDL in Pichia pastoris. The activity of the recombinant protein carrying the C-terminal HIS-tag could not be detected, whereas the purification of the protein carrying the N-terminal HIS-tag yielded moderate amounts (88 mU mL−1) of the partly degraded recombinant protein (Figure 1, lanes 2 and 3). In an effort to increase the yi (...truncated)