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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, 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.
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f2: 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.

Mentions: Human, bovine, zebrafish and arabidopsis TrpRSs were purified following expression in E. coli (Supplementary Fig. S1). We compared the angiostatic activities of full-length and mini forms of human, bovine, and zebrafish TrpRSs, and arabidopsis TrpRS, as assessed by the ability to inhibit HUVEC migration. As shown in Fig. 2, VEGF alone induced HUVEC chemotaxis. Next, we exposed VEGF-stimulated HUVEC cells to human mini TrpRS, and observed that it inhibited VEGF-induced migration (Fig. 2). In contrast, no inhibition of chemotaxis was observed with human full-length TrpRS (Fig. 2). These results are consistent with our previous results5. Moreover, we observed that bovine mini TrpRS inhibited VEGF-stimulated HUVEC chemotaxis to a similar extent as human mini TrpRS, whereas zebrafish mini TrpRS and arabidopsis TrpRS did not (Fig. 2). In addition, the bovine full-length TrpRS and zebrafish full-length TrpRS had no effect on VEGF-stimulated HUVEC chemotaxis (Fig. 2). These results show that bovine, but not zebrafish, TrpRS can function as an angiostatic factor after removal of the extra domain from the full-length TrpRS.


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

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

f2: 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.
Mentions: Human, bovine, zebrafish and arabidopsis TrpRSs were purified following expression in E. coli (Supplementary Fig. S1). We compared the angiostatic activities of full-length and mini forms of human, bovine, and zebrafish TrpRSs, and arabidopsis TrpRS, as assessed by the ability to inhibit HUVEC migration. As shown in Fig. 2, VEGF alone induced HUVEC chemotaxis. Next, we exposed VEGF-stimulated HUVEC cells to human mini TrpRS, and observed that it inhibited VEGF-induced migration (Fig. 2). In contrast, no inhibition of chemotaxis was observed with human full-length TrpRS (Fig. 2). These results are consistent with our previous results5. Moreover, we observed that bovine mini TrpRS inhibited VEGF-stimulated HUVEC chemotaxis to a similar extent as human mini TrpRS, whereas zebrafish mini TrpRS and arabidopsis TrpRS did not (Fig. 2). In addition, the bovine full-length TrpRS and zebrafish full-length TrpRS had no effect on VEGF-stimulated HUVEC chemotaxis (Fig. 2). These results show that bovine, but not zebrafish, TrpRS can function as an angiostatic factor after removal of the extra domain from the full-length TrpRS.

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.