Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B
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
-
Crystal structure of platelet factor 4 complexed with fondaparinuxProtein Data Bank, 4R9W.
-
Drosophila Robo IG1-2 (monoclinic form)Protein Data Bank, 2VRA.
-
CRYSTAL STRUCTURE OF A TERNARY FGF1-FGFR2-HEPARIN COMPLEXProtein Data Bank, 1E0O.
-
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.
Reviewing Editor
- Pamela J Bjorkman, California Institute of Technology, United States
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).
Version history
- Received: March 14, 2019
- Accepted: August 21, 2019
- Accepted Manuscript published: August 22, 2019 (version 1)
- Version of Record published: September 12, 2019 (version 2)
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.
Metrics
-
- 3,050
- views
-
- 515
- downloads
-
- 35
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Numerous lipids are heterogeneously distributed among organelles. Most lipid trafficking between organelles is achieved by a group of lipid transfer proteins (LTPs) that carry lipids using their hydrophobic cavities. The human genome encodes many intracellular LTPs responsible for lipid trafficking and the function of many LTPs in defining cellular lipid levels and distributions is unclear. Here, we created a gene knockout library targeting 90 intracellular LTPs and performed whole-cell lipidomics analysis. This analysis confirmed known lipid disturbances and identified new ones caused by the loss of LTPs. Among these, we found major sphingolipid imbalances in ORP9 and ORP11 knockout cells, two proteins of previously unknown function in sphingolipid metabolism. ORP9 and ORP11 form a heterodimer to localize at the ER-trans-Golgi membrane contact sites, where the dimer exchanges phosphatidylserine (PS) for phosphatidylinositol-4-phosphate (PI(4)P) between the two organelles. Consequently, loss of either protein causes phospholipid imbalances in the Golgi apparatus that result in lowered sphingomyelin synthesis at this organelle. Overall, our LTP knockout library toolbox identifies various proteins in control of cellular lipid levels, including the ORP9-ORP11 heterodimer, which exchanges PS and PI(4)P at the ER-Golgi membrane contact site as a critical step in sphingomyelin synthesis in the Golgi apparatus.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0–6.3 nm) than the MAD within typical helices (4.3–5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.