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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.


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.
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f1: 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.

Mentions: Vertebrate TrpRSs have an NH2-terminal appended domain. In normal cells, human TrpRS exists in two forms: the major full-length protein form and a less abundant mini TrpRS, in which the extra NH2-terminal domain is deleted due to alternative splicing of the pre-mRNA such that Met48 becomes the NH2-terminal residue89 (Fig. 1). We previously found that human mini, but not full-length, TrpRS functions as an angiostatic factor5. Full-length TrpRS (a.a. 1–471) is cleaved by elastase to produce T1 TrpRS (a.a. 71–471) and T2 TrpRS (a.a. 94–471), which also act as angiostatic factors510. Whereas full-length, mini and T1 TrpRSs retain aminoacylation activity, T2 TrpRS is inactive for aminoacylation10.


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)

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

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

f1: 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.
Mentions: Vertebrate TrpRSs have an NH2-terminal appended domain. In normal cells, human TrpRS exists in two forms: the major full-length protein form and a less abundant mini TrpRS, in which the extra NH2-terminal domain is deleted due to alternative splicing of the pre-mRNA such that Met48 becomes the NH2-terminal residue89 (Fig. 1). We previously found that human mini, but not full-length, TrpRS functions as an angiostatic factor5. Full-length TrpRS (a.a. 1–471) is cleaved by elastase to produce T1 TrpRS (a.a. 71–471) and T2 TrpRS (a.a. 94–471), which also act as angiostatic factors510. Whereas full-length, mini and T1 TrpRSs retain aminoacylation activity, T2 TrpRS is inactive for aminoacylation10.

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.