The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin’s cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.
A biomimetic sensor has been developed, that allows for quantification of autoantibodies related to the antiphospholipid syndrome (APS). Autoantibodies directed against the β(2)-glycoprotein-I (β(2)GP-I) are known as the best markers for diagnosis of APS, however, detection of such antibodies is still a challenge. The epitopes of β(2)GP-I are exposed upon binding to negatively charged membranes. The surface of the sensor chips was therefore modified with such type of membranes, on which β(2)GP-I molecules were subsequently immobilized as recognition elements. Using the label-free method, reflectometric interference spectroscopy, it was possible to quantify anti-β(2)GP-I antibodies and to calibrate the sensor chip in buffer. A mild regeneration procedure allows for many consecutive measurements without stripping off the membrane in between.
Patients with hereditary or acquired haemophilia A may develop inhibitory factor VIII (FVIII) antibodies. These disrupt FVIII activity predominantly by preventing the formation of the tenase complex, leading to a serious bleeding disorder. Antibodies without inhibiting activity, however, can also be found when screening patients with haemophilia A under FVIII supplementation. Therefore, the detection of only these allo- or autoantibodies from plasma is not sufficient. Rather, the characterization of the antibody-induced effects on the coagulation cascade should be considered due to its great diagnostic importance. Currently, inhibitory activities are detected by the functional Bethesda assay, which directly measures the delay in clotting time by the patient plasma. However, this assay does not provide information on the cause of the inhibition. Here, we report the development of a surface plasmon resonance (SPR) biosensor that has the potential to integrate both quantitative and functional information on patient antibody characteristics in one measurement. Recombinant FVIII protein was immobilized on the sensor surface to detect antibodies from patient plasma. The interaction of the FIX- and FXa-clotting proteins with the formed anti-FVIII/FVIII complex could be detected subsequently within the same SPR measurement cycle. Inhibitory antibodies led to the prevention of these interactions. Thus, discrimination between the clinically relevant inhibitory and non-inhibitory antibodies was enabled. In a group of 16 patients with inhibitory antibodies (both ELISA- and Bethesda-positive), 5 patients with non-inhibitory antibodies (ELISA-positive but Bethesda-negative) and 12 healthy controls, diagnostic sensitivity and specificity data of 100% for the FIX interaction were achieved using this biomimetic biosensor approach. The new method allows for detection and quantification, as well as for evaluation of inhibitory activity of allo- and autoantibodies, using small sample volume and short analysis time.
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