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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.


Analysis of the interaction of TrpRS with human VE-cadherin by co-immunoprecipitation assays.(A) VE-cadherin binding assay of human T2, mini WT TrpRS, and zebrafish mini WT TrpRS. (B) VE-cadherin binding assay of human mini K114Q, K153Q, K418Q, and E451Q TrpRS mutants. (C) VE-cadherin binding assay of human mini WT TrpRS, zebrafish mini WT TrpRS, zebrafish mini Q107K and Q146K TrpRS mutants. Rabbit polyclonal antibodies against TrpRS were used for Western blot analyses of panels A and B. For panel C, mouse monoclonal antibody against six histidine residues was used. Molecular size markers (in kilodaltons) are shown at the right. A band corresponding to TrpRS or protein G derived from protein G plus-agarose is marked with an arrow or asterisk, respectively.
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f5: Analysis of the interaction of TrpRS with human VE-cadherin by co-immunoprecipitation assays.(A) VE-cadherin binding assay of human T2, mini WT TrpRS, and zebrafish mini WT TrpRS. (B) VE-cadherin binding assay of human mini K114Q, K153Q, K418Q, and E451Q TrpRS mutants. (C) VE-cadherin binding assay of human mini WT TrpRS, zebrafish mini WT TrpRS, zebrafish mini Q107K and Q146K TrpRS mutants. Rabbit polyclonal antibodies against TrpRS were used for Western blot analyses of panels A and B. For panel C, mouse monoclonal antibody against six histidine residues was used. Molecular size markers (in kilodaltons) are shown at the right. A band corresponding to TrpRS or protein G derived from protein G plus-agarose is marked with an arrow or asterisk, respectively.

Mentions: Immunoprecipitation experiments using a recombinant human VE-cadherin Fc chimera, comprising the extracellular domain (EC1-EC5) of human VE-cadherin fused to the Fc domain of human IgG, revealed that human mini WT TrpRS binds to the extracellular domain of VE-cadherin, as does the human T2 TrpRS (Fig. 5A). These data are consistent with previous findings1314. In contrast, the zebrafish mini WT TrpRS did not bind to VE-cadherin (Fig. 5A). Next we performed immunoprecipitation experiments using the human mini TrpRS mutants to identify the residues of human mini TrpRS crucial for binding to VE-cadherin. As shown in Fig. 5B, human mini K114Q, K418Q, and E451Q TrpRS mutants bound to VE-cadherin, whereas the human mini K153Q TrpRS mutant did not. These data suggest that the Lys153 residue of human mini TrpRS is crucial for the interaction with VE-cadherin. Furthermore, the zebrafish mini Q146K TrpRS mutant interacted with VE-cadherin significantly as did human mini WT TrpRS (Fig. 5C). Taken together, we conclude that Lys153 of human mini TrpRS is a VE-cadherin binding site.


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)

Analysis of the interaction of TrpRS with human VE-cadherin by co-immunoprecipitation assays.(A) VE-cadherin binding assay of human T2, mini WT TrpRS, and zebrafish mini WT TrpRS. (B) VE-cadherin binding assay of human mini K114Q, K153Q, K418Q, and E451Q TrpRS mutants. (C) VE-cadherin binding assay of human mini WT TrpRS, zebrafish mini WT TrpRS, zebrafish mini Q107K and Q146K TrpRS mutants. Rabbit polyclonal antibodies against TrpRS were used for Western blot analyses of panels A and B. For panel C, mouse monoclonal antibody against six histidine residues was used. Molecular size markers (in kilodaltons) are shown at the right. A band corresponding to TrpRS or protein G derived from protein G plus-agarose is marked with an arrow or asterisk, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Analysis of the interaction of TrpRS with human VE-cadherin by co-immunoprecipitation assays.(A) VE-cadherin binding assay of human T2, mini WT TrpRS, and zebrafish mini WT TrpRS. (B) VE-cadherin binding assay of human mini K114Q, K153Q, K418Q, and E451Q TrpRS mutants. (C) VE-cadherin binding assay of human mini WT TrpRS, zebrafish mini WT TrpRS, zebrafish mini Q107K and Q146K TrpRS mutants. Rabbit polyclonal antibodies against TrpRS were used for Western blot analyses of panels A and B. For panel C, mouse monoclonal antibody against six histidine residues was used. Molecular size markers (in kilodaltons) are shown at the right. A band corresponding to TrpRS or protein G derived from protein G plus-agarose is marked with an arrow or asterisk, respectively.
Mentions: Immunoprecipitation experiments using a recombinant human VE-cadherin Fc chimera, comprising the extracellular domain (EC1-EC5) of human VE-cadherin fused to the Fc domain of human IgG, revealed that human mini WT TrpRS binds to the extracellular domain of VE-cadherin, as does the human T2 TrpRS (Fig. 5A). These data are consistent with previous findings1314. In contrast, the zebrafish mini WT TrpRS did not bind to VE-cadherin (Fig. 5A). Next we performed immunoprecipitation experiments using the human mini TrpRS mutants to identify the residues of human mini TrpRS crucial for binding to VE-cadherin. As shown in Fig. 5B, human mini K114Q, K418Q, and E451Q TrpRS mutants bound to VE-cadherin, whereas the human mini K153Q TrpRS mutant did not. These data suggest that the Lys153 residue of human mini TrpRS is crucial for the interaction with VE-cadherin. Furthermore, the zebrafish mini Q146K TrpRS mutant interacted with VE-cadherin significantly as did human mini WT TrpRS (Fig. 5C). Taken together, we conclude that Lys153 of human mini TrpRS is a VE-cadherin binding site.

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.