Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution

Abstract

Human tryptophanyl-tRNA synthetase (TrpRS) exists in two forms: a full-length TrpRS and a mini TrpRS. We previously found that human mini, but not full-length, TrpRS is an angiostatic factor. Moreover, it was shown that the interaction between mini TrpRS and the extracellular domain of vascular endothelial (VE)-cadherin is crucial for its angiostatic activity. However, the molecular mechanism of the angiostatic activity of human mini TrpRS is only partly understood. In the present study, we investigated the effects of truncated (mini) form of TrpRS proteins from human, bovine, or zebrafish on vascular endothelial growth factor (VEGF)-stimulated chemotaxis of human umbilical vein endothelial cells (HUVECs). We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not. Next, to identify residues crucial for the angiostatic activity of human mini TrpRS, we prepared several site-directed mutants based on amino acid sequence alignments among TrpRSs from various species and demonstrated that a human mini K153Q TrpRS mutant cannot inhibit VEGF-stimulated HUVEC migration and cannot bind to the extracellular domain of VE-cadherin. Taken together, we conclude that the Lys153 residue of human mini TrpRS is a VE-cadherin binding site and is therefore crucial for its angiostatic activity.

Figure 1. Schematic representation of human, bovine, zebrafish and arabidopsis TrpRS constructs used in this study.

Sequence alignments of full-length (FL) and mini TrpRS proteins are depicted schematically. Numbers on the left and right correspond to the NH₂- and COOH-terminal residues, respectively. The open boxes indicate the core catalytic domain conserved among eukaryotic TrpRSs and the shaded boxes represent the NH₂-terminal appended domains specific to vertebrate TrpRSs.

Figure 2. Effects of human, bovine, zebrafish and arabidopsis TrpRSs on VEGF-induced endothelial migration.

VEGF (0.5 nM) and full-length (FL) or mini TrpRS (500 nM) were used. Migrating cells were counted in four random fields (×100 total magnification) per insert and were averaged. All data are expressed as means ± SEM from at least four independent experiments. Data were analyzed by one-way ANOVA followed by Tukey-Kramer post hoc tests. **P < 0.01 versus the VEGF control.

Figure 3. Effects of human mini WT, K114Q, K153Q, K418Q, and E451Q TrpRSs on VEGF-induced endothelial migration.

VEGF (0.5 nM) and mini TrpRS (500 nM) were used. Migrating cells were counted in four random fields (×100 total magnification) per insert and were averaged. All data are expressed as means ± SEM from at least four independent experiments. Data were analyzed by one-way ANOVA followed by Tukey-Kramer post hoc tests. **P < 0.01 compared to the VEGF plus human mini WT TrpRS.

Figure 4. Effects of zebrafish mini WT, Q107K, Q146K, Q411K, and H445E TrpRSs on VEGF-induced endothelial migration.

VEGF (0.5 nM) and mini TrpRS (500 nM) were used. Migrating cells were counted in four random fields (×100 total magnification) per insert and were averaged. All data are expressed as means ± SEM from at least four independent experiments. Data were analyzed by one-way ANOVA followed by Tukey-Kramer post hoc tests. *P < 0.05, **P < 0.01 compared to the VEGF plus zebrafish mini WT TrpRS.

Figure 5. Analysis of the interaction of TrpRS with human VE-cadherin by co-immunoprecipitation assays.

(A) VE-cadherin binding assay of human T2, mini WT TrpRS, and zebrafish mini WT TrpRS. (B) VE-cadherin binding assay of human mini K114Q, K153Q, K418Q, and E451Q TrpRS mutants. (C) VE-cadherin binding assay of human mini WT TrpRS, zebrafish mini WT TrpRS, zebrafish mini Q107K and Q146K TrpRS mutants. Rabbit polyclonal antibodies against TrpRS were used for Western blot analyses of panels A and B. For panel C, mouse monoclonal antibody against six histidine residues was used. Molecular size markers (in kilodaltons) are shown at the right. A band corresponding to TrpRS or protein G derived from protein G plus-agarose is marked with an arrow or asterisk, respectively.

Figure 6. Aminoacylation activity of human and zebrafish TrpRSs toward yeast tRNA^Trp.

Aminoacylation efficiencies were determined from initial rates and calculated as pmol/min of aminoacylated tRNA^Trp synthesized during a 1 min incubation. The assays included 200 nM TrpRS and 500 μM yeast tRNA. Values represent the means ± standard deviation from four experiments. (A) Aminoacylation activity of human mini WT, K114Q, K153Q, K418Q, and E451Q TrpRSs. (B) Aminoacylation activity of zebrafish mini WT, Q107K, Q146K, Q411K, and H445E TrpRSs.

Figure 7. A residue of human TrpRS crucial for angiostatic activity.

(A) Comparison of amino acid residues at the positions tested for angiostatic activity in this study among mammalian, bird, reptilian, amphibian, and fish TrpRS proteins. Multiple sequence alignment was performed by Clustal W with manual adjustments. Conserved crucial acidic (Glu or Asp) and basic (Arg or Lys) residues are highlighted in yellow. (B) Tertiary structural conformation of Lys153 and the NH₂-terminal appended domain of human full-length TrpRS, and tryptophanyl-adenylate (Trp-AMP) bound to human full-length TrpRS (Protein Data Bank code: 1R6T). Lys153, NH₂-terminal appended domain, and Trp-AMP are indicated in red, blue and green, respectively. (C) A molecular docking model of the complex between human mini TrpRS (Protein Data Bank code: 1ULH) and EC1-EC2 of VE-cadherin (Protein Data Bank code: 3PPE). Lys153 and 382–389 resides in human mini TrpRS are indicated in red and black, respectively. Human mini TrpRS and EC1-EC2 of VE-cadherin are highlighted in yellow and purple, respectively. NH₂-terminal Trp2 and Trp4 residues of VE-cadherin are represented by purple space-filling balls.