Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
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Figures 1, 4, 5 and Extended Data Figs. 1, 7, and 8 have associated raw data (uncropped blots and/or gel pictures) in Supplementary Fig. 1. Figures 1, 2 and Extended Data Figs. 1, 4 have associated raw data (Excel files) for all bar graphs representing data from experiments involving mouse models. For the molecular modelling of palmitoyl-CoA docking into GS, models and trajectories are available on Figshare (doi: 10.6084/m9.figshare.6575438). Any additional information required to interpret, replicate or build upon the Methods or findings reported in the manuscript is available from the corresponding author upon request.
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We acknowledge R. Levine for supplying purified bacterial GS; L. Van Den Bosch and W. Scheveneels for providing primary mouse astrocytes; S. Trenson, I. Crèvecoeur, S. Noppen, L. Van Berckelaer and R. Van Berwaer for technical assistance; S.-M. Fendt, D. Verdegem and C. Ulens for discussions and suggestions; W. Vermaelen, A. Acosta Sanchez, A. Brajic, A. Sobrino and M. Cockx for experimental assistance; L.-C. Conradi and A. Pircher for supplying materials; and E. Wauters, A. Wolthuis and J. Jaekers for providing tissues for EC isolations. HecBioSim and PRACE are acknowledged for allocation of computer time. J.G., A.R.C., C.D., C.L., J.K., F.M.-R., S.R. and S.Va. are supported by the FWO; A.Z. by LE&RN/FDRS; B.C. by IWT; U.B. by a Marie Curie-IEF Fellowship; H.H. by an EMBO Long-Term Fellowship; J.D.v.B. by a LSBR fellowship; R.Cu. by a British Heart Foundation Intermediate Clinical Fellowship; and X.W. by the American Cancer Society RSG, NIH/NCI and NIH/NIDDK. F.C., G.S. and F.L.G. are supported by the EPSRC; X.L. by the State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center at Sun Yat-Sen University, and the National Natural Science Foundation of China (81330021 and 81670855). P.C. is supported by a Federal Government Belgium grant, long-term structural Methusalem funding by the Flemish Government, a Concerted Research Activities Belgium grant, grants from the FWO, Foundation against Cancer and ERC Advanced Research Grant. G.E. and M.Dew. received a Foundation against Cancer grant and G.E. received a FWO ‘Krediet aan navorsers’.