Distribution of variants identified in NEU1.

<p>Schematic representation of NEU1 protein, with amino acid position on the X axis. Black vertical lines divide the 6 exons of <i>NEU1</i> gene. Line graph indicates the conservation score for each amino acid position as calculated by ScoreCons. The identified missense variants are represented as squares, with Y values corresponding to PolyPhen score. The missense variants already identified as pathological alleles in sialidosis are represented as diagonal crosses. Below the graph, schematic representation of NEU1 structural elements: full color blocks represent beta-strand elements, while crossed blocks represent helices. Color code identifies the 6 blades of the β-propeller structure as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104229#pone.0104229-Chavas1" target="_blank">[35]</a>. Gray boxes represent the Asp-box elements and essential catalytic residues are indicated with dots and their residue number. Positions of all elements are relative to the amino acid position in the graph above.</p

Distribution of SNVs in secondary structural elements of NEU1 protein.

<p>Numbers in brackets represent the p-value associated to the enrichment of SNVs in the corresponding category calculated as described in Methods. Classification of variants: mis, missense; syn, synonymous.</p

Subcellular localization study of V217A and D234N NEU1 mutant proteins.

<p>Confocal microscopy images showing the subcellular distribution of NEU1 wild-type, V217A and D234N mutants. NEU1 proteins were detected using specific rabbit anti-NEU1 antibody and revealed with Alexa-555 secondary antibody. Wild-type NEU1 labeling resulted in a vesicular pattern (A), mainly colocalizing with the lysosomal marker LAMP1 detected with mouse anti-LAMP1 and revealed with Alexa-405 secondary antibody (B, merge in C). The V217A (D) and D234N (G) mutant proteins showed a tubulo-reticular localization. Almost no colocalization between V217A and D234N mutants and LAMP1 (E–F and H–I) could be detected. Insets represent enlargement of the indicated areas.</p

Distribution of SNVs in the exon of <i>NEU1</i> gene.

<p>Based on <i>NEU1</i> cDNA sequence NM_000434.3. Numbers in brackets represent the p-value associated to the enrichment of SNVs in the corresponding exon calculated as described in Methods. Classification of variants: mis, missense; syn, synonymous; UTR, located in the untranslated regions.</p

Missense and LoF variants in <i>NEU1</i> CDS.

<p>*  = variants selected for in vitro functional studies;</p>†<p> = variants already annotated as pathological mutations in HGMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104229#pone.0104229-Stenson1" target="_blank">[13]</a>.</p><p>The two missense variants identified as putative pathogenic mutations and the frameshift variant are shown in bold. Impact prediction summarizes results from the PolyPhen2, SIFT and VEP tools: D, damaging; B, benign or tolerated. GERP (Genomic Evolutionary Rate Profiling) score measures the level of conservation for the indicated nucleotide (see Methods). MAF, minor allele frequency; nd, not reported in the corresponding database. <i>NEU1</i> cDNA sequence refers to NM_000434.3, protein sequence refers to NP_000425.1.</p

Summary information on <i>NEU1</i> gene and related SNVs.

<p><i>NEU1</i> cDNA sequence refers to NM_000434.3, protein sequence refers to NP_000425.1. LoF, loss of function; UTR, located in the untranslated regions. dN/dS measures the level of evolutionary constrain acting on the <i>NEU1</i> gene (see Methods).</p

Sialidase activity assay and immunoblotting analysis.

<p>(A) Sialidase specific activity calculated for COS7 cells non transfected (NT), transfected with NEU1 wt alone (NEU1), PPCA alone (PPCA), NEU1 wt and PPCA (WT) and NEU1 mutants and PPCA. V217A and D234N mutant proteins resulted in a significant reduction in sialidase activity (p<0.05, marked with *). (B) Immunoblotting for PPCA and NEU1 proteins in each sample. NEU1 showed the expected signals between 40 and 46 kDa, corresponding to different glycosylation states; PPCA showed the expected signal at 32 kDa, corresponding to the heavy peptide of the active form of the protein. α-tub was used for protein loading normalization. The image is representative of 3 replicate experiments.</p

NEU1 structure analysis and localization of the 9 candidate missense mutations.

<p>Top (A) and lateral (B) views of the structural model of NEU1 protein. The two amino acids V217 and D234, identified in this work as affected by new putative pathological mutations, are shown in red; the 7 amino acids affected by the other candidate missense variants tested are shown in green. Other amino acids already reported as mutated in sialidosis and likely involved in NEU1-PPCA interaction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104229#pone.0104229-Lukong1" target="_blank">[28]</a> are shown in blue. The black arrow in (B) indicates the side of the catalytic crevice. (C) Detailed view of the localization of V217 and D234. These two amino acids, together with G218, L231, W240, G243 and A298 define a region of the NEU1 protein that could represent an important site in the surface interaction with PPCA partner.</p

Molecular cloning and biochemical characterization of sialidases from zebrafish (Danio rerio)

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