Molecular cloning and biochemical characterization of sialidases from zebrafish

Sialidases remove sialic acid residues from glycoconjugates. In mammals four different sialidases exist: NEU1 is located in the lysosomes, NEU2 is a cytosolic protein, whereas NEU3 and NEU4 are associated to the plasma membrane and intracellular membranous structures, respectively. Functional aspects of each sialidase are not fully understood, but sialoglyconjugates alterations are associated with cancer. NEU3 modifies the cell ganglioside content, also through cell-to-cell interaction. A marked increase of NEU3 characterizes colon and renal cancers compared with adjacent non-tumor cells. Moreover, NEU4 influences the invasive properties of colon cancers. We thus decided to study this group of glycohydrolases in zebrafish. A zebrafish genome-wide search allowed us to detect 11 genes related to human sialidases: neu1 and neuf are putative orthologs of NEU1 and NEU4, respectively, while the remaining genes are more closely related to NEU3. All gene transcripts studied but neud are detected by RT-PCR, and in situ hybridizations show a localized expression pattern in gut and lens for neua and neuf, respectively. Expression of neua, neub, neuc and neuf in mammalian cells demonstrates they encode active sialidase enzymes. Neua, Neuc, Neuf are membrane-associated and show very acidic pH optima, below 3.0, whereas Neub is a soluble sialidase with a pH optimum of 5.6. Transfection experiments demonstrate that the membrane-associated enzymes modify the ganglioside pattern of living COS7 cells, consistent with the results obtained with NEU3 of mammalian origin. We plan to test the developmental roles of these zebrafish sialidases in order to clarify their biological roles in vertebrates.