New insights on the sialidase protein family revealed by a phylogenetic analysis in metazoa

Abstract

Sialidases are glycohydrolytic enzymes present from virus to mammals that remove sialic acid from oligosaccharide chains. Four different sialidase forms are known in vertebrates: the lysosomal NEU1, the cytosolic NEU2 and the membrane-associated NEU3 and NEU4. These enzymes modulate the cell sialic acid content and are involved in several cellular processes and pathological conditions. Molecular defects in NEU1 are responsible for sialidosis, an inherited disease characterized by lysosomal storage disorder and neurodegeneration. The studies on the biology of sialic acids and sialyltransferases, the anabolic counterparts of sialidases, have revealed a complex picture with more than 50 sialic acid variants selectively present in the different branches of the tree of life. The gain/loss of specific sialoconjugates have been proposed as key events in the evolution of deuterostomes and Homo sapiens, as well as in the host-pathogen interactions. To date, less attention has been paid to the evolution of sialidases. Thus we have conducted a survey on the state of the sialidase family in metazoan. Using an in silico approach, we identified and characterized sialidase orthologs from 21 different organisms distributed among the evolutionary tree: Metazoa relative (Monosiga brevicollis), early Deuterostomia, precursor of Chordata and Vertebrata (teleost fishes, amphibians, reptiles, avians and early and recent mammals). We were able to reconstruct the evolution of the sialidase protein family from the ancestral sialidase NEU1 and identify a new form of the enzyme, NEU5, representing an intermediate step in the evolution leading to the modern NEU3, NEU4 and NEU2. Our study provides new insights on the mechanisms that shaped the substrate specificity and other peculiar properties of the modern mammalian sialidases. Moreover, we further confirm findings on the catalytic residues and identified enzyme loop portions that behave as rapidly diverging regions and may be involved in the evolution of specific properties of sialidases.

Figure 1. Analysis of sialidase catalytic crevice.

(A) The superimposition of the structural models obtained for sialidase enzymes showed that the localization of the 6 key residues (those essentials for catalysis) within the protein structure is well conserved. The enzyme competitive inhibitor DANA is depicted in red. (B) Surface representation of the catalytic crevice in human NEU2 structure. Key residues, in magenta, are located near the C1 carboxyl group and the C2 (hemiketal carbon of Sias involved in the formation of the sialosyl linkage), while other residues interacting with the substrate, in blue, interact with the C-4 hydroxyl group, C-5 acetamido group and glycerol side-chain of DANA, where most of the natural Sia modifications occur. (C) Analysis of the 5 amino acid residues interacting with the competitive inhibitor DANA but not essential in the catalytic process, as identified from human NEU2 crystal structure. Subfamily LOGO analysis comparing NEU1 subgroup with other sialidase subgroups revealed specific differences on these 5 residues. X indicate missing residues and significant differences are marked with *. L217 shows no subgroup specific conservation in this comparison. (D) LOGO representations of the multiple alignments of the five sialidase subgroups show high sequence conservation for all the 6 key residues involved in the catalytic process. Yellow arrows in (C) and (D) indicate the considered residues. Residues are numbered according to human NEU2 protein sequence. The same color code is used in panels (B), (C) and (D) to indicate the residues that coordinate DANA.

Figure 2. Schematic representation of the sialidase structure.

β-sheets are represented as grey arrows, loops are depicted as black lines and the positions of the 6 key residues involved in the catalysis are indicated with yellow dots. The five Asp-boxes sites (Asp 1–5) are indicated with a green square. LOGOs representing sequence conservation in each one of the 5 sialidase subgroups are reported for each Asp-box. The black crossed boxes (1–6) indicate the loops in which large functional domains have been identified in leech, bacterial or viral sialidases. Number legend: 1: M. decora; 2: C. perfrigens, S. pneumoniae; 3: V. cholerae; 4: C. perfringens, Newcastle virus, M. decora; 5: S. tiphimurium; 6: influenza B and influenza N9 viruses. The red crossed box indicates the highly variable loop identified in vertebrates, surrounded by two highly conserved sequence blocks, represented by blue diamonds. For these blocks LOGOs representation of the multiple alignments in the 5 sialidase subgroups are also reported.

Figure 3. Length of the highly variable loop connecting the first and second strand of the fifth blade of the β-propeller.

Length of the highly variable loop connecting the first and second strand of the fifth blade of the β-propeller, identified in sialidases from vertebrates (red crossed box in Figure 2). The loop is considerably shorter in NEU1 and shows the greater length variability in NEU3 and NEU4 enzymes.

Figure 4. Analysis of the 9 loops emerging on the same side of the active site.

(A) Horizontal bar chart representation of the mean length of the single loops in the 5 sialidase subgroups. Significant differences in length occur in Loop 3, 4 and 6 and are indicated with *. (B) Top view of the human NEU2 structure showing the 9 loops analyzed. Loops with significant length variations are near the glycerol portion of DANA and are represented in red, other loops are in blue. Catalytic key residues are represented as sticks and DANA inhibitor is colored in magenta. (C) Structural representation of Loop 3, containing the E111 residue, in different sialidases. Comparison of structural models revealed that this loop is considerably shorter in NEU1s than in other sialidases. The short α-helix structure containing E111 residue is absent in NEU1s. The positions of each loop within the human NEU2 structure are: Loop 0 (aa 16–18), Loop 1 (aa 15–18), Loop 2 (aa 42–49), Loop 3 (aa 107–122), Loop 4 (aa 183–190), Loop 5 (aa 238–243), Loop 6 (aa 261–274), Loop 7 (aa 299–306), Loop 8 (aa 329–332), Loop 9 (aa 357–359).

Figure 5. Analysis of NEU1 specific features.

Multiple alignment of NEU1 protein sequences revealed that the signal peptide present at the N-terminal in the mammalian enzymes is poorly conserved in vertebrates (A), while the lysosomal localization signal at C-terminus is still present throughout Chordata (B). The conserved aromatic residue, usually placed at −4 position, is indicated by the yellow arrow in (B). (C) LOGO representation of the 4 residues essential for the interaction between NEU1 and PPCA. These residues are highly conserved in the NEU1 subgroup but they are lost in other sialidases. Residue numbering is according to human NEU1 sequence.

Figure 6. Exon structure analysis.

Exon structure of the four already known sialidase subgroups (NEU1–4; grey bars), of the newly identified NEU5 sialidases (green bars), of the 5 sialidases from M. brevicollis (light blue bars) and of the NEU1 sialidases from hemichordate S. kowalevskii (yellow bars). Horizontal bars represent the protein sequence and the position of the exon junctions within the polypeptides are indicated by the black lines. Dotted black lines in NEU3 subgroup indicate additional exons found only in the avian species analyzed (G. gallus, T. guttata), while in NEU4 indicate an additional exon find only in X. tropicalis. The bar representation of the protein sequences is not in scale with the exon length, but the reciprocal position of exon junctions in different subgroups are respected.

Figure 7. Conservation of phosphorylatable residues.

Heatmap representation of the level of conservation of the 84 predicted phosphorylation sites is given for the global set of 83 sialidases and for each one of the five sialidase subgroup in (A) and for the 4 taxonomical groups in (B). Phosphorilatyon sites are identifeid according to their absolute position in the global alignment of the 83 sialidase sequences. Globally (A) and specific subgroup (B) sites showing statistical significant conservation (p<0.01) are indicated: * corresponds to sequence conservation ≥60%; ** corresponds to conservation ≥90%.

Figure 8. Complete phylogenetic tree of the sialidase gene family.

Complete phylogenetic tree of the sialidase gene family reconstructed using Maximum Likelihood method and WAG+G amino acid substitution model. The tree is drawn to scale, with branch lengths measured as number of substitutions per site. The tree with the highest log likelihood (-38941.9521) is shown. Human sialidases are reported in bold. Different subgroups are represented with different colors: NEU1 in green, NEU2 in purple, NEU3 in cyan, NEU4 in red and NEU5 in blue. Branches containing the protist K. brevis and the Choanoflagellata M. brevicollis are represented with grey lines. Bootstrap analysis was conducted with 1000 replicates (bootstrap values are reported in detailed trees in Figure 9). Sialidase from the flagellate S. vortens (Svo NEU1) is placed at the tree root. The tree was constructed, visualized and manipulated using MEGA5.

Figure 9. NEU1 and NEU2–5 phylogenetic subtrees in sialidase protein family.

Detailed view of the phylogenetic subtrees of NEU1 sialidase subgroup (A) and of the other subgroups (B). The newly identified NEU5 subgroup emerges at the root of the NEU2, 3 and 4 subclasses. Bootstrap values are indicated for every node. Color code is used for different subclasses: NEU from protist K. brevis and from choanoflagellate M. brevicollis in grey, NEU1 in green, NEU2 in purple, NEU3 in cyan, NEU4 in red, NEU5 in blue. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.

Figure 10. Co-evolution of the sialidase (NEU) and sialyltransferase (ST) gene families reconstructed using Mesquite software.

Each subgroups in the two families is represented by a line of different color, NEUs on the left side, STs on the right side. The development of new classes of glycoconjugates in Echinodermata corresponds to the evolution of the NEU5 enzyme in sialidases. The appearance of the complete set of STs in Mammalia corresponds to the appearance of NEU2 in sialidase family. ST line pointing to Choanoflagellata is dotted since we were unable to retrieve any STs related sequence in Choanoflagellata and thus the actual presence of STs in this taxon can not be confirmed. Sialyltransferases are named according to .