Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution.
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.
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Terumasa
T
Nakamoto
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Miki
M
Miyanokoshi
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Tomoaki
T
Tanaka
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Keisuke
K
Wakasugi
D019943:Amino Acid Substitution; D000596:Amino Acids; D049148:Aminoacylation; D000818:Animals; D001665:Binding Sites; D015820:Cadherins / Q000378:metabolism; D002633:Chemotaxis; D042783:Endothelial Cells / Q000378:metabolism; D004789:Enzyme Activation; D019143:Evolution, Molecular; D006801:Humans; D011485:Protein Binding; D014369:Tryptophan-tRNA Ligase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D000596:Amino Acids; D015820:Cadherins; D042461:Vascular Endothelial Growth Factor A; D014369:Tryptophan-tRNA Ligase