HIV-1 Tat and Heparan Sulfate Proteoglycans Orchestrate the Setup of in Cis and in Trans Cell-Surface Interactions Functional to Lymphocyte Trans-Endothelial Migration

HIV-1 transactivating factor Tat is released by infected cells. Extracellular Tat homodimerizes and engages several receptors, including integrins, vascular endothelial growth factor receptor 2 (VEGFR2) and heparan sulfate proteoglycan (HSPG) syndecan-1 expressed on various cells. By means of experimental cell models recapitulating the processes of lymphocyte trans-endothelial migration, here, we demonstrate that upon association with syndecan-1 expressed on lymphocytes, Tat triggers simultaneously the in cis activation of lymphocytes themselves and the in trans activation of endothelial cells (ECs). This “two-way” activation eventually induces lymphocyte adhesion and spreading onto the substrate and vascular endothelial (VE)-cadherin reorganization at the EC junctions, with consequent endothelial permeabilization, leading to an increased extravasation of Tat-presenting lymphocytes. By means of a panel of biochemical activation assays and specific synthetic inhibitors, we demonstrate that during the above-mentioned processes, syndecan-1, integrins, FAK, src and ERK1/2 engagement and activation are needed in the lymphocytes, while VEGFR2, integrin, src and ERK1/2 are needed in the endothelium. In conclusion, the Tat/syndecan-1 complex plays a central role in orchestrating the setup of the various in cis and in trans multimeric complexes at the EC/lymphocyte interface. Thus, by means of computational molecular modelling, docking and dynamics, we also provide a characterization at an atomic level of the binding modes of the Tat/heparin interaction, with heparin herein used as a structural analogue of the heparan sulfate chains of syndecan-1.

Mapping the Structural and Dynamic Determinants of pH-sensitive Heparin Binding to Granulocyte Macrophage-colony Stimulating Factor

GMCSF is an immunomodulatory cytokine that is harnessed as a therapeutic. GMCSF is known to interact with other clinically important molecules, such as heparin, suggesting that endogenous and administered GMCSF has the potential to modulate orthogonal treatment outcomes. Thus, molecular level characterization of GMCSF and its interactions with other biologically active compounds is critical to understanding these mechanisms and predicting clinical outcomes. Here, we dissect the molecular motions and structural contributions that facilitate the GMCSF-heparin interaction, previously shown to be pH-dependent, using NMR spectroscopy, SPR, and molecular docking. We find that GMCSF and heparin binding is related to a change in flexibility reflected in the dynamic profile of GMCSF at acidic pH. The molecular motions driving this interaction largely occur on the ms-µs timescale. Interestingly, we find that GMCSF and heparin binding is not only pH-dependent but is also heparin chain length dependent. We propose a mechanism where a positive binding pocket that is not fully solvent accessible at neutral pH becomes more accessible at acidic pH, allowing heparin to dock with the protein.

Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake

Cancer-derived small extracellular vesicles (sEVs) induce stromal cells to become permissive for tumor growth. However, it is unclear whether this induction solely occurs through transfer of vesicular cargo into recipient cells. Here we show that cancer-derived sEVs can stimulate endothelial cell migration and tube formation independently of uptake. These responses were mediated by the 189 amino acid isoform of vascular endothelial growth factor (VEGF) on the surface of sEVs. Unlike other common VEGF isoforms, VEGF189 preferentially localized to sEVs through its high affinity for heparin. Interaction of VEGF189 with the surface of sEVs profoundly increased ligand half-life and reduced its recognition by the therapeutic VEGF antibody bevacizumab. sEV-associated VEGF (sEV-VEGF) stimulated tumor xenograft growth but was not neutralized by bevacizumab. Furthermore, high levels of sEV-VEGF were associated with disease progression in bevacizumab-treated cancer patients, raising the possibility that resistance to bevacizumab might stem in part from elevated levels of sEV-VEGF.

Langerin-Heparin Interaction: Analysis of the Binding to the Non-Lectin Site

Langerin is a C-type lectin involved in the immune response that forms a trimer in its active form. It can interact with carbohydrates using 2 sites with different selectivity, the C-lectin site, a Ca2+-mediated binding, and the cleft between chains. Here we report the complementary analysis of the interaction between a heparin-like hexasaccharide 1 and langerin at the second site.