Nanodiscs (ND) are lipid bilayer membrane patches held by amphiphilic scaffolding proteins (MSP) of ~10 nm in diameter. Nanodiscs have been developed as lipid nanoplatforms for structural and functional studies of membrane and membrane associated proteins. Their size and monodispersity have rendered them unique for electron microscopy (EM) and single particle analysis studies of proteins and complexes either spanning or associated to the ND membrane. Binding of blood coagulation factors and complexes, such as the Factor VIII (FVIII) and the Factor VIIIa - Factor IXa (intrinsic tenase) complex to the negatively charged activated platelet membrane is required for normal haemostasis. In this study we present our work on optimising ND, specifically designed to bind FVIII at close to physiological conditions. The binding of FVIII to the negatively charged ND rich in phosphatidylserine (PS) was followed by electron microscopy at three different PS compositions and two different membrane scaffolding protein (MSP1D1) to lipid ratios. Our results show that the ND with highest PS content (80 %) and lowest MSP1D1 to lipid ratio (1:47) are the most suitable for structure determination of the membrane-bound FVIII by single particle EM. Our preliminary FVIII 3D reconstruction as bound to PS containing ND demonstrates the suitability of the optimised ND for structural studies by EM. Further assembly of the activated FVIII form (FVIIIa) and the whole FVIIIa-FIXa complex on ND, followed by EM and single particle reconstruction will help to identify the protein-protein and protein-membrane interfaces critical for the intrinsic tenase complex assembly and function.
Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2. Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A - a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7. In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described. The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.
We present a methodology of lipid nanotubes (LNT) and nanodisks technologies optimized in our laboratory for structural studies of membrane-associated proteins at close to physiological conditions. The application of these lipid nanotechnologies for structure determination by cryoelectron microscopy (cryo-EM) is fundamental for understanding and modulating their function. The LNTs in our studies are single bilayer galactosylceramide based nanotubes of ~20 nm inner diameter and a few microns in length, that self-assemble in aqueous solutions. The lipid nanodisks (NDs) are self-assembled discoid lipid bilayers of ~10 nm diameter, which are stabilized in aqueous solutions by a belt of amphipathic helical scaffold proteins. By combining LNT and ND technologies, we can examine structurally how the membrane curvature and lipid composition modulates the function of the membrane-associated proteins. As proof of principle, we have engineered these lipid nanotechnologies to mimic the activated platelet's phosphtaidylserine rich membrane and have successfully assembled functional membrane-bound coagulation factor VIII in vitro for structure determination by cryo-EM. The macromolecular organization of the proteins bound to ND and LNT are further defined by fitting the known atomic structures within the calculated three-dimensional maps. The combination of LNT and ND technologies offers a means to control the design and assembly of a wide range of functional membrane-associated proteins and complexes for structural studies by cryo-EM. The presented results confirm the suitability of the developed methodology for studying the functional structure of membrane-associated proteins, such as the coagulation factors, at a close to physiological environment.
Membrane-bound Factor VIII (FVIII) has a critical function in blood coagulation as the pro-cofactor to the serine-protease Factor IXa (FIXa) in the FVIIIa-FIXa complex assembled on the activated platelet membrane. Defects or deficiency of FVIII cause Hemophilia A, a mild to severe bleeding disorder. Despite existing crystal structures for FVIII, its membrane-bound organization has not been resolved. Here we present the dimeric FVIII membrane-bound structure when bound to lipid nanotubes, as determined by cryo-electron microscopy. By combining the structural information obtained from helical reconstruction and single particle subtomogram averaging at intermediate resolution (15-20 Å), we show unambiguously that FVIII forms dimers on lipid nanotubes. We also demonstrate that the organization of the FVIII membrane-bound domains is consistently different from the crystal structure in solution. The presented results are a critical step towards understanding the mechanism of the FVIIIa-FIXa complex assembly on the activated platelet surface in the propagation phase of blood coagulation.
Blood hemostasis is a delicate balance between plasma proteins (coagulation factors) forming active membrane-bound complexes. Understanding their assembly is fundamental for the cure of blood disorders, such as hemophilia and thrombosis. To this goal we design suitable lipid nano-platforms supporting coagulation complexes and allowing extensive structure-functional studies required for successful nano-drug design. We aim to study the membrane-bound organization of Factor VIII (FVIII) forms, as organized onto lipid nanotubes (LNT) by combining Cryo-electron microscopy (Cryo-EM) with biophysical methods. Here we present our preliminary Cryo-EM results of the membrane-bound organization of two recombinant FVIII forms: human full length (hrFVIII-FL) and porcine -B domain deleted (prFVIII-BDD) at different lipid composition. Cryo-electron micrographs of A, B. hrFVIII-FL and C, D. prFVIII-BDD organized onto LNT at low and high phsophatedylcholine (PS) concentration, respectively. Scale bar 100 nm.
Purpose: Microparticles (MP) are non-biological inorganic bacterial-sized particles (0.1-0.7mm) from endogenous formation (calcium phosphate) or dietary intake (AlSi and Ti02), which may contribute to the pathogenesis of inflammatory bowel disease (IBD). This study was to establish the method for MP detection in human intestine. Methods: Biopsy tissue samples were collected from 5 IBD and 5 cancer screening patients undergoing colonoscopy.Tissue sections were cut at variable thickness (5, 10 and 20 um) prior to analysis on the Very Sensitive Elemental and Structural Probe Employing Radiation from a Synchrotron (VESPERS) microprobe beamline at the Canadian Light Source. They were mapped with micro-X-ray fluorescence spectroscopy to determine the spatial location of the MPs at an X-ray excitation energy of 13 keV. The beam had a spot size of 4 mm with an 8 mm step-size and 15 second dwell time. Tested sample conditions include formalin fixation, section thickness, mounting substrate (regular glass slide and Lexan film), and embedding medium (OCT). Results: Randomly selected areas in the mucosa or submucosa were observed. There was significant contamination in formalin-fixed tissue samples causing non-specific background. VESPERS exhibited greatest sensitivity with tissue sections of 20 um in thickness and demonstrated the best signal versus noise ratio. Specific signals of MPs from samples on Lexan film were much better with relatively lower background than those on glass slide. The OCT was free of contamination. Conclusion: Synchrotron-based X-ray microprobe techniques can be used for detecting and mapping MPs in normal and IBD affected human intestine. This will allow us further elucidation of the immunopathological role of MPs in IBD patients. Frozen fresh tissue sections of 20 um thickness mounted on Lexan film is the superior method of sample processing.
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