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

Nakamoto T, Miyanokoshi M, Tanaka T, Wakasugi K - Sci Rep (2016)

Bottom Line: We previously found that human mini, but not full-length, TrpRS is an angiostatic factor.We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.

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.

No MeSH data available.


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.
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f3: 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.

Mentions: Next, to identify crucial residues for the angiostatic activity of human mini TrpRS, we performed a sequence alignment between mammalian and fish TrpRS proteins and pinpointed key differences between mammalian and fish TrpRS sequences, with a particular focus on exposed residues with positive or negative charges (Supplementary Fig. S2). We identified Lys114, Lys153, Lys418, and Glu451 of human TrpRS, which correspond to Gln107, Gln146, Gln411, and His445 of zebrafish TrpRS, respectively, as potential candidate residues, and prepared the site-directed human mini TrpRS mutants, K114Q, K153Q, K418Q, and E451Q, and the zebrafish mini TrpRS mutants, Q107K, Q146K, Q411K, and H445E, with numbering based on the residue numbers of full-length TrpRS. We examined the effects of TrpRS mutants on VEGF-induced cell migration. As shown in Fig. 3, we observed that the human mini K153Q TrpRS mutant did not inhibit VEGF-stimulated HUVEC chemotaxis, whereas the human mini K114Q, K418Q, and E451Q TrpRS mutants inhibited VEGF-stimulated HUVEC migration as did the human mini WT TrpRS. In addition, we show that the zebrafish mini Q146K TrpRS mutant, in which Gln146 (corresponding to Lys153 of human TrpRS) was converted to Lys, significantly inhibited VEGF-stimulated HUVEC migration (Fig. 4). Taken together, these data suggest that Lys153 of human mini TrpRS is crucial for its angiostatic activity.


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

Nakamoto T, Miyanokoshi M, Tanaka T, Wakasugi K - Sci Rep (2016)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4837363&req=5

f3: 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.
Mentions: Next, to identify crucial residues for the angiostatic activity of human mini TrpRS, we performed a sequence alignment between mammalian and fish TrpRS proteins and pinpointed key differences between mammalian and fish TrpRS sequences, with a particular focus on exposed residues with positive or negative charges (Supplementary Fig. S2). We identified Lys114, Lys153, Lys418, and Glu451 of human TrpRS, which correspond to Gln107, Gln146, Gln411, and His445 of zebrafish TrpRS, respectively, as potential candidate residues, and prepared the site-directed human mini TrpRS mutants, K114Q, K153Q, K418Q, and E451Q, and the zebrafish mini TrpRS mutants, Q107K, Q146K, Q411K, and H445E, with numbering based on the residue numbers of full-length TrpRS. We examined the effects of TrpRS mutants on VEGF-induced cell migration. As shown in Fig. 3, we observed that the human mini K153Q TrpRS mutant did not inhibit VEGF-stimulated HUVEC chemotaxis, whereas the human mini K114Q, K418Q, and E451Q TrpRS mutants inhibited VEGF-stimulated HUVEC migration as did the human mini WT TrpRS. In addition, we show that the zebrafish mini Q146K TrpRS mutant, in which Gln146 (corresponding to Lys153 of human TrpRS) was converted to Lys, significantly inhibited VEGF-stimulated HUVEC migration (Fig. 4). Taken together, these data suggest that Lys153 of human mini TrpRS is crucial for its angiostatic activity.

Bottom Line: We previously found that human mini, but not full-length, TrpRS is an angiostatic factor.We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.

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.

No MeSH data available.