Phosphotyrosine phosphatase R3 receptors: Origin, evolution and structural diversification

<div><p>Subtype R3 phosphotyrosine phosphatase receptors (R3 RPTPs) are single-spanning membrane proteins characterized by a unique modular composition of extracellular fibronectin repeats and a single cytoplasmatic protein tyrosine phosphatase (PTP) domain. Vertebrate R3 RPTPs consist of five members: PTPRB, PTPRJ, PTPRH and PTPRO, which dephosphorylate tyrosine residues, and PTPRQ, which dephosphorylates phophoinositides. R3 RPTPs are considered novel therapeutic targets in several pathologies such as ear diseases, nephrotic syndromes and cancer. R3 RPTP vertebrate receptors, as well as their known invertebrate counterparts from animal models: PTP52F, PTP10D and PTP4e from the fruitfly <i>Drosophila melanogaster</i> and F44G4.8/DEP-1 from the nematode <i>Caenorhabditis elegans</i>, participate in the regulation of cellular activities including cell growth and differentiation. Despite sharing structural and functional properties, the evolutionary relationships between vertebrate and invertebrate R3 RPTPs are not fully understood. Here we gathered R3 RPTPs from organisms covering a broad evolutionary distance, annotated their structure and analyzed their phylogenetic relationships. We show that R3 RPTPs (i) have probably originated in the common ancestor of animals (metazoans), (ii) are variants of a single ancestral gene in protostomes (arthropods, annelids and nematodes); (iii) a likely duplication of this ancestral gene in invertebrate deuterostomes (echinodermes, hemichordates and tunicates) generated the precursors of PTPRQ and PTPRB genes, and (iv) R3 RPTP groups are monophyletic in vertebrates and have specific conserved structural characteristics. These findings could have implications for the interpretation of past studies and provide a framework for future studies and functional analysis of this important family of proteins.</p></div

Protein and DNA trees showing the evolutionary relationships between RPTPs.

<p>A) Bayes protein concatenated tree (see text for description of model parameters and analysis specifics). Values at the nodes in the tree indicate the Bayesian posterior probability for that node. B) DNA elide Bayes tree (see text for description of model parameters and analysis specifics). Values at the nodes in the tree indicate the Bayesian posterior probability for that node. Protein and DNA parsimony, and additional Bayesian phylogenetic trees are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172887#pone.0172887.s006" target="_blank">S2 File</a>.</p

Invertebrate and vertebrate PTP domain similarity.

<p>Heat map representing color-coded blastp E value of PTP domains of R3 PTPRs from vertebrates and inverebrates deuterostomes, protostomes and sponges. Note the strong similarity of the sponge PTP sequence with PTPRF and PTPRG. E values are coloured from green (low similarity) to red (high similarity) (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172887#pone.0172887.s008" target="_blank">S1 Table</a> for numerical E values; 100% similarity corresponds to an E value of 0.0). Full scientific names of species are: Co, <i>Capsaspora owczarzaki</i>; Aq, <i>Amphimedon queenslandica</i>; Ct, <i>Capitella teleta</i>; Dm, <i>Drosophila melanogaster</i>; Ce, <i>Caenorhabditis elegans</i>; Hc, <i>Haemonchus contortus</i>; Sp, <i>Strongylocentrotus purpuratus</i>; Sk, <i>Saccoglossus kowalevskii</i>; Cs, <i>Ciona Savigny</i>; Ci, <i>Ciona intestinalis</i>; Hs, <i>Homo sapiens</i>. Other sequences Chicken (Gg) <i>Gallus gallus</i>; Fish (Dr) <i>Danio rerio</i>.</p

Domain architecture of human R3 RPTP members.

<p>Schematic representation of human R3 subtype RPTP protein members. For catalytic PTP, FN3 and signal peptide symbols see the figure. Black and light blue boxes represent the transmembrane segments and the cytoplasmatic regions after the PTP domain, respectively. Note the larger size of the juxtamembrane FN3 domain in PTPRQ, PTPRB and PTPRJ proteins [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172887#pone.0172887.ref015" target="_blank">15</a>]. Protein amino acid numbers are indicated in parenthesis below the protein names.</p

Conservation of UPK2/3 uroplakins intron/exon pattern and cysteine pairs in vertebrates.

<p>The alignment of the full-length sequences of human UPK3a, UPK3b, UPK3c, UPK2a, lizard UPK2b and zebrafish UPK3d was generated by the MAFFT server with default parameters, except for the position of intron one, which was manually curated and aligned. Introns 1 to 5 are indicated above the sequences. The amino acids split by the introns are shaded in blue and red according to phase 1 and 2, respectively. Conserved cysteines are shaded in yellow, and numbered 1 to 4 from the N-terminus. Note that UPK3d has two additional cysteines (blue box) and that C3–C4 in UPK2a and C1–C2 in UPK3c are not conserved in all the organisms (for an alignment of a full vertebrate set of UPK2/3 proteins see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170196#pone.0170196.s002" target="_blank">S2 Fig</a>). The signal peptide, transmembrane domain and cytoplasmic tail are enclosed in blue, black and red dashed boxes, respectively. The amino acids in the transmembrane helices are underlined. Note that although the sequences of UPK2/3 members are only weakly similar, their intron/exon conjunctions and several key cysteine residues are highly conserved, strengthening their shared evolutionary origin. Amino acid identity: identical (*); strongly similar (:); weakly similar (.).</p

Evolutionary relationship between UPK3 and PTPRQ genes.

<p>The evolution of the similarity region in a broad spectrum of species spanning sponge to mammals was analyzed using a combined protein sequence and molecular morphology matrix (see text for details). Bayesian phylogenetic analysis was used to generate a tree with posterior probabilities given at the nodes in the tree (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170196#sec002" target="_blank">Materials and Methods</a> section for details of Bayesian analysis). <i>Monosiga</i> R5 PTPRF was used as outgroup since R5 PTPRF is the closest to R3 PTPR.</p

Similarity between the cytoplasmic tails of UPK3a and the <i>Ciona</i> R3 PTPR sequences without a catalytic domain (CionaNocat.1 and CionaNocat.2).

<p>Upper panel: cartoon of UPK3a and either one of the <i>Ciona</i> sequences with no catalytic domain (symbols are the same as in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170196#pone.0170196.g002" target="_blank">Fig 2</a>). Lower panel: alignment of UPK3a, UPK3b, CionaNocat.1 and CionaNocat.2 cytoplasmic tail sequences. Code for shaded regions: residues identical and similar to the CionaNocat.1 sequence are highlighted in green and yellow, respectively. Predicted phosphoritable tyrosine residues are colour in magenta. The frog tyrosine (Tyr249), which is transiently phosphorylated upon gamete fertilization, and the human threonine (Thr244), which is phosphorylated upon adhesion of uropathogenic <i>E</i>. <i>coli</i> to the urothelium, are marked with vertical arrows and Thr/Ser244 are coloured in blue. Intron 5 position just after the TMD domain is marked by a vertical arrow.</p

Shared region between UPK2/3 uroplakins and human PTPRQ, PTPRB and PTPRJ R3 PTPR members.

<p>Symbols include: long FN3-like juxtamembrane domain exons, pink boxes; transmembrane domain, black box; numbers below the boxes indicate the number of amino acid residues; introns (vertical arrows numbered 1 to 5); blue and red color denote phase 1 (between the 1st and 2nd positions) and phase 2 (2nd and 3rd); star above the human UPK3a sequence indicates that although only human UPK3a sequence is shown, all UPK2/3 human proteins share the same intron/exon pattern with the exception of exon 5, which is absent in UPK2a and UPK2b (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170196#pone.0170196.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170196#pone.0170196.s002" target="_blank">S2 Fig</a>); C1–C4 indicate the four cysteines of UPK3a conserved in PTPRQ and PTPRB, but not in PTPRJ. Note the absence of the long FN3-like juxtamembrane domain in PTPRH and PTPRO (dotted line). See box for additional symbols for signal peptides, catalytic PTP domains and FN3 domains (number indicated). The size of the proteins is indicated in parenthesis below the protein names.</p

Characteristics of the long FN3-like juxtamembrane region in vertebrate and invertebrate R3 PTPR sequences.

<p>The long FN3-like domain of UPK3a, UPK3b, PTPRQ and PTPRB from representative vertebrates and R3 PTPR-related sequences from representative invertebrates were aligned using the MAFFT server with default parameters and printed out with BoxShade (identical and similar residues are represented by different grey shadings). The positions of the introns 1 to 4 are marked and the amino acids split between two exons (phase 0, 1 and 2) are shaded in green, blue and red, respectively; cysteine residues 1 to 4 are shaded in yellow; putative furin cleavage sites are marked with vertical red bars between the cleaved amino acids and high conserved short signature sequences are underlined in red numbered 1 to 7. The sequences cover vertebrates (black); tunicates (<i>Ciona</i>, blue); hemichordates (acorn worm, green); echinoderms (sea urchin, orange); lophotrochozoa (annelid, light brown); edquisozoan (nematode, blue and fruit fly, magenta); and porifera (sponge, purple). Organisms’ scientific names are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170196#pone.0170196.s006" target="_blank">S1 File</a>. PTPRB, UPK3b sequences were removed from the alignment and are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170196#pone.0170196.s004" target="_blank">S4 Fig</a> along with UPK3a and PTPRQ sequences for comparison. UPK2 sequences were not included in the analysis since they lack the C3–C4 sequence region.</p

Homo- and Heterobinuclear Cu²⁺ and Zn²⁺ Complexes of Ditopic Aza Scorpiand Ligands as Superoxide Dismutase Mimics

Two polytopic aza-scorpiand-like ligands, 6-[7-(diaminoethyl)-3,7-diazaheptyl]-3,6,9-triaza-1-(2,6-pyridina)­cyclodecaphane (<b>L1</b>) and 6-[6′-[3,6,9-triaza-1-(2,6-pyridina)­cyclodecaphan-6-yl]-3-azahexyl]-3,6,9-triaza-1-(2,6-pyridina)­cyclodecaphane (<b>L2</b>), have been synthesized. The acid–base behavior and Cu²⁺, Zn²⁺, and Cu²⁺/Zn²⁺ mixed coordination have been analyzed by potentiometry, cyclic voltammetry, and UV–vis spectroscopy. The resolution of the crystal structures of [Cu₂<b>L2</b>Cl₂]­(ClO₄)₂·1.67H₂O (<b>1</b>), [Cu₂H<b>L2</b>Br₂]­(ClO₄)₃·1.5H₂O (<b>2</b>), and [CuZn<b>L2</b>Cl₂]­(ClO₄)₂·1.64H₂O (<b>3</b>) shows, in agreement with the solution data, the formation of homobinuclear Cu²⁺/Cu²⁺ and heterobinuclear Cu²⁺/Zn²⁺ complexes. The metal ions are coordinated within the two macrocyclic cavities of the ligand with the involvement of a secondary amino group of the bridge in the case of <b>1</b> and <b>3</b>. Energy-dispersive X-ray spectroscopy confirms the 1:1 Cu²⁺/Zn²⁺ stoichiometry of <b>3</b>. The superoxide dismutase (SOD) activities of the Cu²⁺/Cu²⁺ and Cu²⁺/Zn²⁺ complexes of <b>L1</b> and <b>L2</b> have been evaluated using nitro blue tetrazolium assays at pH 7.4. The IC₅₀ and <i>k</i>_cat values obtained for the [Cu₂<b>L1</b>]⁴⁺ complex rank among the best values reported in the literature for Cu-SOD mimics. Interestingly, the binuclear Cu²⁺ complexes of <b>L1</b> and <b>L2</b> have low toxicity in cultures of mammalian cell lines and show significant antioxidant activity in a copper-dependent SOD (SOD1)-defective yeast model. The results are rationalized by taking into account the binding modes of the Cu²⁺ ions in the different complexes