title: Molecular cloning and biochemical characterization of sialidases from zebrafish (Danio rerio).
abstract: Sialidases remove sialic acid residues from various sialo-derivatives. To gain further insights into the biological roles of sialidases in vertebrates, we exploited zebrafish (Danio rerio) as an animal model. A zebrafish transcriptome- and genome-wide search using the sequences of the human NEU polypeptides as templates revealed the presence of seven different genes related to human sialidases. neu1 and neu4 are the putative orthologues of the mammalian sialidases NEU1 and NEU4 respectively. Interestingly, the remaining genes are organized in clusters located on chromosome 21 and are all more closely related to mammalian sialidase NEU3. They were thus named neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5. Using RT-PCR (reverse transcription-PCR) we detected transcripts for all genes, apart from neu3.4, and whole-mount in situ hybridization experiments show a localized expression pattern in gut and lens for neu3.1 and neu4 respectively. Transfection experiments in COS7 (monkey kidney) cells demonstrate that Neu3.1, Neu3.2, Neu3.3 and Neu4 zebrafish proteins are sialidase enzymes. Neu3.1, Neu3.3 and Neu4 are membrane-associated and show a very acidic pH optimum below 3.0, whereas Neu3.2 is a soluble sialidase with a pH optimum of 5.6. These results were further confirmed by subcellular localization studies carried out using immunofluorescence. Moreover, expression in COS7 cells of these novel zebrafish sialidases (with the exception of Neu3.2) induces a significant modification of the ganglioside pattern, consistent with the results obtained with membrane-associated mammalian sialidases. Overall, the redundancy of sialidases together with their expression profile and their activity exerted on gangliosides of living cells indicate the biological relevance of this class of enzymes in zebrafish.
authors: Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.
authors: Marta
authors: M
authors: Manzoni
authors: Paolo
authors: P
authors: Colombi
authors: Nadia
authors: N
authors: Papini
authors: Luana
authors: L
authors: Rubaga
authors: Natascia
authors: N
authors: Tiso
authors: Augusto
authors: A
authors: Preti
authors: Bruno
authors: B
authors: Venerando
authors: Guido
authors: G
authors: Tettamanti
authors: Roberto
authors: R
authors: Bresciani
authors: Francesco
authors: F
authors: Argenton
authors: Giuseppe
authors: G
authors: Borsani
authors: Eugenio
authors: E
authors: Monti
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000201:enzymology; D015027:Zebrafish
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D009439:Neuraminidase
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