The genome sequence of the protostome Daphnia pulex encodes respective orthologues of a neurotrophin, a Trk and a p75NTR: evolution of neurotrophin signaling components and related proteins in the bilateria

Abstract

Background: Neurotrophins and their Trk and p75NTR receptors play an important role in the nervous system. To date, neurotrophins, Trk and p75NTR have only been found concomitantly in deuterostomes. In protostomes, homologues to either neurotrophin, Trk or p75NTR are reported but their phylogenetic relationship to deuterostome neurotrophin signaling components is unclear. Drosophila has neurotrophin homologues called Spätzles (Spz), some of which were recently renamed neurotrophins, but direct proof that these are deuterostome neurotrophin orthologues is lacking. Trks belong to the receptor tyrosine kinase (RTK) family and among RTKs, Trks and RORs are closest related. Flies lack Trks but have ROR and ROR-related proteins called NRKs playing a neurotrophic role. Mollusks have so far the most similar proteins to Trks (Lymnaea Trk and Aplysia Trkl) but the exact phylogenetic relationship of mollusk Trks to each other and to vertebrate Trks is unknown. p75NTR belongs to the tumor necrosis factor receptor (TNFR) superfamily. The divergence of the TNFR families in vertebrates has been suggested to parallel the emergence of the adaptive immune system. Only one TNFR representative, the Drosophila Wengen, has been found in protostomes. To clarify the evolution of neurotrophin signaling components in bilateria, this work analyzes the genome of the crustacean Daphnia pulex as well as new genetic data from protostomes.

Results: The Daphnia genome encodes a neurotrophin, p75NTR and Trk orthologue together with Trkl, ROR, and NRK-RTKs. Drosophila Spz1, 2, 3, 5, 6 orthologues as well as two new groups of Spz proteins (Spz7 and 8) are also found in the Daphnia genome. Searching genbank and the genomes of Capitella, Helobdella and Lottia reveals neurotrophin signaling components in other protostomes.

Conclusion: It appears that a neurotrophin, Trk and p75NTR existed at the protostome/deuterostome split. In protostomes, a "neurotrophin superfamily" includes Spzs and neurotrophins which respectively form two paralogous families. Trks and Trkl proteins also form closely related paralogous families within the protostomian RTKs, whereby Trkls are absent in deuterostomes. The finding of p75NTR in several protostomes suggests that death domain TNFR superfamily proteins appeared early in evolution.

Figure 1

Phylogenetic tree of Neurotrophin/Spz genes: Spz family. The full Spz/neurotrophin(Nt) tree is shown on the left side, while on the right side, an enlarged view of the Spz family is depicted. BI phylogenetic tree is shown, but analyses with ML are also represented via bootstrap support values on the tree. BI pp (posterior probability) values figure directly above the branches while ML bootstrap values are directly below the branches. Fruit fly: Drosophila melanogaster, Moquitoe Aa: Aedes aegypti, Mosquitoe Ag: Anopheles gambiae, Dappu: Daphnia pulex, Salmon louse: Lepeophtheirus salmonis, Sea louse: Caligus rogercresseyi, Brine shrimp: Artemia franciscana, Fl. prawn: Penaeus chinensis, Am. Lobster: Homarus americanus, Louse: Pediculus humanus corporis, TickIs: Ixodes scapularis, TickRm: Rhipicephalus microplus, Helobdella: Hellobdella robusta, Capitella: Capitella Sp. I, Lottia: Lottia gigantea, Human: Homo sapiens, Frog: Xenopus leavis, Chicken: Gallus gallus, Zebrafish: Danio rerio, Amphioxus: Branchiostoma floridae, Acorn worm: Saccoglossus kowalevskii, Sea urchin: Strongylocentrotus purpuratus.

Figure 2

Phylogenetic analysis of Neurotrophin/Spz genes: Neurotrophin family. The full Spz/neurotrophin tree is shown on the left side, while on the right side, an enlarged view of the neurotrophin family is depicted. The species names and explanation of numerical values along the branches are described in the legend of Figure 1.

Figure 3

Alignment of neurotrophin Cys knots to Spz C-106 sequences. An arrow head (above alignment) indicates where partial Spz6 sequences were removed. I to VI designate conserved "Cys knot" cysteines (white on black). "H" (below alignment) indicates a hydrophobic site. Cys unique to Spz subfamilies are in grey. Highlights indicate frequent characteristic residues of either Spzs (red tones) or neurotrophins (blue-green tones). Canonical invertebrate deuterostome neurotrophins (amphioxus and sea urchin NT) are separated from those belonging to the 2^nd sea urchin neurotrophin "NT2" and the two acorn worm ESTs ("NTa" and "NTb"). Dmagna: Daphnia magna. Dappu: Daphnia pulex.

Figure 4

Alignment of the C-terminal sequence of Daphnia pulex Spz7 and 8 to arthropod Spz2s. Alignment of the Cys knot sequence and C-terminal of one respective representative of Daphnia pulex Spz7 (Dappu-Spz7A) and Spz8 (Dappu-Spz8A) to Spz2 sequences of Daphnia (Dappu-Spz2), Aedes (Aa: Aedes aegypti) and Drosophila (Dm: Drosophila melanogaster). Conserved Cys forming the Cys knot are in white on black background and are numbered below from I to VI. Conserved residues between the sequences are indicated below by an asterisk (*). Arrowheads indicate Cys (also highlighted in grey) characteristically conserved in both Spz7 and Spz8, or only conserved in Spz8. The unique C-terminal extension of Spz2 proteins is present in Dappu-Spz2 as well as a conserved "NY(D/N)YHPIIDFF" sequence motif (in white on black) and these are absent in Spz7 and Spz8 representatives.

Figure 5

The sequence of Daphnia pulex neurotrophin. A. Predicted amino acid sequence of Dappu-NT, the Daphnia pulex neurotrophin. The signal peptide is underlined and the putative propeptide cleavage site is marked by an arrow head. Conserved Cysteines for the characteristic Cys knot are in white against a black background. B. Alignment of Dappu-NT C-terminus to other neurotrophin C-termini. Identical residues are in white over a grey background. The 5 Cys (white on black background) of the neurotrophin Cys-knot are conserved. Most residues from the vertebrate neurotrophin consensus motif " [GSRE]-C- [KRL]-G- [LIVT]- [DE]-x(3)- [YW]-x-S-x-C" are also conserved (underlined). Hs: Homo sapiens, Bf: Branchiostoma floridae, Sp: Strongylocentrotus purpuratus, Sk: Saccoglossus kowalevskii.

Figure 6

Predicted Intron-Exon organization of the Daphnia pulex neurotrophin gene. Exons are represented by boxes and the number of nucleotides included within each exon is indicated above the boxes. Introns figure as a line and their length in number of nucleotides is indicated below. The type of sequence encoded by each exon is shown by initials and colour codes. SP (Dotted fill): Signal Peptide; PP (Grey): ProPeptide; Cys knot (white): CI to CVI stand for the six cysteines of the Cysteine knot. CI to CVI figure on the respective exons encoding them.

Figure 7

Daphnia pulex Trk amino acid sequence. Amino acid positions are numbered on the left. The putative signal sequence cleavage site is indicated by an arrowhead. Leucine-rich motifs are in italics and flanking cysteine clusters in bold. Both Ig-like domains are underlined; conserved asparagines with structural roles for ligand binding are in grey and double underlined. The transmembrane region is underlined by a thick line. The first phosphorylation site by cAMP/cGMP-dependent kinase proteins, RYS is shown with a black background, as is the tyrosine responsible for Shc recruitment. Within the tyrosine kinase domain (boxed), the lysine responsible for ATP binding and the second phosphorylation site by cAMP/cGMP-dependent kinase proteins, RKFT, are shown by a black background. The autophosphorylation sequence (DIYSSDYYK) is highlighted in grey and the autophosphorylated tyrosines are shown on a black background. The tyrosine responsible for PLC gamma docking is underlined and in larger font.

Figure 8

Genomic organization of the Daphnia Trk relative to the vertebrate and amphioxus Trks. The diagram shows the part of the modular Trk protein encoded by each exon. The upper part of the diagram shows the different domains of the protein while the three diagrams below show the exon organization relative to these domains in vertebrates, amphioxus (Invertebrate deuterostome) and Daphnia (Protostome). SP: Signal Peptide; LRR: Leucine rich repeats; Cys rich: Cysteine rich domain; IG: Immunoglobulin domain; eJM: External Juxta-Membrane domain; TM: Transmembrane domain; Shc binding: Shc binding site; Tyrosine kinase: Tyrosine kinase domain; PLCg: Phospholipase C-γ docking site. Intron exon junctions are represented by vertical lines across the rectangle that represents each protein-coding sequence. Conserved intron-exon junctions between any invertebrate species and vertebrates are in red.

Figure 9

Phylogenetic analysis of RTKs. Trks, Trkls, NRKs and RORs are analyzed together with Drosophila DTrk (DmDTrk) as an outgroup. A: Phylogenetic tree based on full length sequences: A BI derived tree topology is shown as well as support values for the nodes computed by BI (pp: posterior probability directly above the branches) and ML (ML bootstrap values directly below the branches). B: Phylogenetic tree based on sequences encoding the tyrosine kinase domains only. The topology and support values are depicted in the same way as in A. Human (Homo sapiens), Chicken (Gallus gallus), Zebrafish (Danio rerio), Amphioxus (Branchiostoma floridae), Sea urchin (Strongylocentrotus purpuratus), Lottia (Lottia gigantean), Aplysia (Aplysia californica), Dappu-(Daphnia pulex), Capitella (Capitella Sp. I), Honey Bee (Apis mellifera), Pea Aphid (Acyrthosiphon pisum), Jewel wasp (Nasonia vitripennis), Drosophila (Drosophila melanogaster).

Figure 10

Alignment of Trkls. Dappu- (Daphnia pulex), Bee (Apis mellifera), Pea Aphid (Acyrthosiphon pisum), Jewel wasp (Nasonia vitripennis), Lottia (Lottia gigantea) and Aplysia (Aplysia californica) trkl sequences are aligned. The EGF domain is underlined and conserved Cys are highlighted. The transmembrane domain is underlined. The Tyr in the NPxY motif prior to the tyrosine kinase domain is highlighted black on white. Important functional residues within the tyrosine kinase domain such as the phosphorylation site YXXDYY are also highlighted.

Figure 11

Alignment of Daphnia ROR to other ROR sequences. Alignment of Daphnia pulex ROR (Dappu-ROR) with human (Homo sapiens (Hs)) ROR1, fly (Drosophila melanogaster (Dm)) ROR and sea urchin (Strongylocentrotus purpuratus (Sp)) ROR. The SpROR N-terminal does not figure in the alignment for space purposes, it contains an N-terminal extension with an additional Immunoglobulin domain (not shown) that does not align to its counterparts. In the alignment, the immunoglobulin domain on the extracellular side is underlined, while the frizzled Cystein Rich Domain (CRD) is in italics with conserved cysteines highlighted in grey. In the extracytoplasmic part, the kringle domain is highlighted in grey. The transmembrane domain is underlined. In the cytoplasmic tyrosine kinase domain of the protein, the YXXDYY that corresponds to the site of phosphorylation within the TrkB activation domain is highlighted in grey. Potential SH2 binding sites are also highlighted.

Figure 12

Schematic representation of Trk-related RTKs. Organization of Trk, Trkl, NRK and ROR protein domains in various protostome and deuterostome representatives: The intracellular tyrosine kinases (TK) are depicted in different shades of red depending on their affiliation to the Trk, Trkl, NRK or ROR families. The transmembrane domains are in grey. The immunoglobulin (Ig) domains are in different shades of blue depending on their type (Ig, IgC1 or IgGC2), the Kringle domains are in dark pink (KR), the EGF domain is in light pink, the Cys Rich Domains are in yellow (C), and leucine rich repeats are in green (LRR).

Figure 13

Alignment of Daphnia pulex p75NTR to other p75NTRs and frog NRH1. Dappu- (Daphnia pulex), Mouse (Mus musculus), Frog (Xenopus leavis), Sea Urchin (Strongylocentrotus purpuratus), Lottia (Lottia gigantea) and Capitella (Capitella Sp. I). Identical residues are marked by an asterisk. Conserved Cysteines in the "Cys Rich Domain" (CRD) that defines TNFRSF members are highlighted in grey. The transmembrane domain is underlined. The death domain is highlighted in grey.