Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B
- University of Birmingham, United Kingdom
- Wellcome Sanger Institute, United Kingdom
- University Hospital Würzburg, Germany
- Université de Strasbourg, France
- Sygnature Discovery Limited, United Kingdom
- University of Helsinki, Finland
- Helsinki University Hospital Research Institute, Finland
- University of Westminster, United Kingdom
- Peak Proteins Limited, United Kingdom
- Wellcome Trust Sanger Institute, United Kingdom
Abstract
The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically-modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.
Data availability
Diffraction data have been deposited in PDB under the accession code 6R0X
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Crystal structure of platelet factor 4 complexed with fondaparinuxProtein Data Bank, 4R9W.
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Drosophila Robo IG1-2 (monoclinic form)Protein Data Bank, 2VRA.
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CRYSTAL STRUCTURE OF A TERNARY FGF1-FGFR2-HEPARIN COMPLEXProtein Data Bank, 1E0O.
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CRYSTAL STRUCTURE OF A TERNARY FGF2-FGFR1-HEPARIN COMPLEXProtein Data Bank, 1FQ9.
Article and author information
Author details
Funding
British Heart Foundation (RG/15/13/31673)
- Jun Mori
- Alexandra Mazharian
- Yotis A Senis
British Heart Foundation (FS/13/1/29894)
- Yotis A Senis
Deutsche Forschungsgemeinschaft (VO 2134-1/1)
- Timo Vögtle
Medical Research Council (Confidence in Concept 2018)
- Timo Vögtle
- Yotis A Senis
Wellcome (206194)
- Gavin J Wright
British Heart Foundation (FS/15/58/31784)
- Alexandra Mazharian
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal procedures were undertaken with the U.K. Home Office approval (project license No P46252127) in accordance with the Animals (Scientific Procedures) Act of 1986.
Human subjects: Blood was collected from healthy drug-free volunteers. Donors gave full informed consent according to the Helsinki declaration. Ethical approval for collecting blood was granted by Birmingham University Internal Ethical Review (ERN_11-0175 and ERN_15-0973).
Copyright
© 2019, Vögtle et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B
eLife 8:e46840.
https://doi.org/10.7554/eLife.46840
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Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.
