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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.


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.
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f7: 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.

Mentions: In the present study, we showed that bovine mini TrpRS inhibited VEGF-stimulated HUVEC chemotaxis to an extent similar to that of human mini TrpRS, whereas zebrafish mini or arabidopsis full-length TrpRS did not. We further demonstrated that Lys153 of human mini TrpRS is crucial for its angiostatic activity but not for its aminoacylation activity. Lys153 is located in the eukaryote-specific patch, which extends from Glu82 to Lys15436. As shown in Fig. 7A, Lys153 is conserved among mammalia, aves, reptillia, and amphibia, but not among osteichtes, implying that the mini TrpRSs from mammalia, aves, reptillia, and amphibia may act as angiostatic factors. Further studies are necessary to investigate protein-protein interactions between TrpRS and VE-cadherin from various species and to examine the angiostatic activities of TrpRSs by using endothelial cells from various species.


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)

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

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

f7: 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.
Mentions: In the present study, we showed that bovine mini TrpRS inhibited VEGF-stimulated HUVEC chemotaxis to an extent similar to that of human mini TrpRS, whereas zebrafish mini or arabidopsis full-length TrpRS did not. We further demonstrated that Lys153 of human mini TrpRS is crucial for its angiostatic activity but not for its aminoacylation activity. Lys153 is located in the eukaryote-specific patch, which extends from Glu82 to Lys15436. As shown in Fig. 7A, Lys153 is conserved among mammalia, aves, reptillia, and amphibia, but not among osteichtes, implying that the mini TrpRSs from mammalia, aves, reptillia, and amphibia may act as angiostatic factors. Further studies are necessary to investigate protein-protein interactions between TrpRS and VE-cadherin from various species and to examine the angiostatic activities of TrpRSs by using endothelial cells from various species.

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.