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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.


Aminoacylation activity of human and zebrafish TrpRSs toward yeast tRNATrp.Aminoacylation efficiencies were determined from initial rates and calculated as pmol/min of aminoacylated tRNATrp 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.
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f6: Aminoacylation activity of human and zebrafish TrpRSs toward yeast tRNATrp.Aminoacylation efficiencies were determined from initial rates and calculated as pmol/min of aminoacylated tRNATrp 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.

Mentions: Because it has been previously reported that the NH2-terminal Trp2 and Trp4 residues of VE-cadherin are docked into the Trp- and adenosine-binding pockets of human TrpRS13, we next investigated the aminoacylation activity of the various TrpRSs. Since previous data showed that eukaryotic TrpRSs can aminoacylate yeast tRNATrp 3132333435, we investigated the aminoacylation of yeast tRNATrp by the recombinant TrpRSs. As shown in Fig. 6A, we observed that the human mini K153Q TrpRS mutant exhibited similar aminoacylation activity as the human mini WT TrpRS, implying that the K153Q mutation has no effect upon the tertiary structure within the Trp- and adenosine-binding pockets. Moreover, the aminoacylation activity of the zebrafish mini Q146K TrpRS mutant was also almost the same as that of the zebrafish mini WT TrpRS (Fig. 6B). Taken together, we conclude that Lys153 of human mini TrpRS is crucial for its angiostatic activity but not for its aminoacylation 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)

Aminoacylation activity of human and zebrafish TrpRSs toward yeast tRNATrp.Aminoacylation efficiencies were determined from initial rates and calculated as pmol/min of aminoacylated tRNATrp 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Aminoacylation activity of human and zebrafish TrpRSs toward yeast tRNATrp.Aminoacylation efficiencies were determined from initial rates and calculated as pmol/min of aminoacylated tRNATrp 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.
Mentions: Because it has been previously reported that the NH2-terminal Trp2 and Trp4 residues of VE-cadherin are docked into the Trp- and adenosine-binding pockets of human TrpRS13, we next investigated the aminoacylation activity of the various TrpRSs. Since previous data showed that eukaryotic TrpRSs can aminoacylate yeast tRNATrp 3132333435, we investigated the aminoacylation of yeast tRNATrp by the recombinant TrpRSs. As shown in Fig. 6A, we observed that the human mini K153Q TrpRS mutant exhibited similar aminoacylation activity as the human mini WT TrpRS, implying that the K153Q mutation has no effect upon the tertiary structure within the Trp- and adenosine-binding pockets. Moreover, the aminoacylation activity of the zebrafish mini Q146K TrpRS mutant was also almost the same as that of the zebrafish mini WT TrpRS (Fig. 6B). Taken together, we conclude that Lys153 of human mini TrpRS is crucial for its angiostatic activity but not for its aminoacylation 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.