Zebrafish encodes several sialidases belonging to the NEU3 group, the plasma membrane-associated member of the family with high specificity toward ganglioside substrates. Neu3.1, Neu3.2 and Neu 3.3 have been expressed in E. coli and purified using the pGEX-2T expression system. Although all the enzymes are expressed by bacterial cells, Neu3.1 formed insoluble aggregates that hampered its purification. Neu3.2 and Neu3.3 formed oligomers as demonstrated by gel filtration chromatography experiments. Actually, the first formed a trimer whereas the second a pentamer. Intriguingly, despite relevant degree of sequence identity and similarity, the two enzymes showed peculiar substrate specificities toward gangliosides other than GM3, two glycoproteins and two forms of sialyllactose. Using molecular modelling and the crystal structure of the human cytosolic sialidase NEU2 as a template, the 3D models of the sialidases from zebrafish have been generated. As expected, the 3D models showed the typical six blade beta-propeller typical of sialidases, with an overall highly conserved active site architecture. The differences among the three zebrafish enzymes and human NEU2 are mainly located in the loops connecting the antiparallel beta strands of the propeller core. These portions of the proteins are probably responsible for the differences observed in substrate specificities, as well as in the different subcellular localization and aggregation features observed in solution. Finally, the in silico analysis of RNA-Seq data evidenced a peculiar expression profile of the three genes during embryogenesis, suggesting different roles of these sialidases during development.
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Dept. of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy.