Supported Lipid Bilayer Platform To Test Inhibitors of the Membrane Attack Complex: Insights into Biomacromolecular Assembly and Regulation

Abstract

Complement activation plays an important role in innate immune defense by triggering formation of the membrane attack complex (MAC), which is a biomacromolecular assembly that exhibits membrane-lytic activity against foreign invaders including various pathogens and biomaterials. Understanding the details of MAC structure and function has been the subject of extensive work involving bulk liposome and erythrocyte assays. However, it is difficult to characterize the mechanism of action of MAC inhibitor drug candidates using the conventional assays. To address this issue, we employ a biomimetic supported lipid bilayer platform to investigate how two MAC inhibitors, vitronectin and clusterin, interfere with MAC assembly in a sequential addition format, as monitored by the quartz crystal microbalance-dissipation (QCM-D) technique. Two experimental strategies based on modular assembly were selected, precincubation of inhibitor and C5b-7 complex before addition to the lipid bilayer or initial addition of inhibitor followed by the C5b-7 complex. The findings indicate that vitronectin inhibits membrane association of C5b-7 via a direct interaction with C5b-7 and via competitive membrane association onto the supported lipid bilayer. On the other hand, clusterin directly interacts with C5b-7 such that C5b-7 is still able to bind to the lipid bilayer, and clusterin affects the subsequent binding of other complement proteins involved in the MAC assembly. Taken together, the findings in this study outline a biomimetic approach based on supported lipid bilayers to explore the interactions between complement proteins and inhibitors, thereby offering insight into MAC assembly and regulation.

Single-molecule kinetics of pore assembly by the membrane attack complex

“…It is likely that C5b6 membrane-binding is dominated by electrostatic interactions of negatively charged lipid headgroups (such as PG lipids commonly found in Gram-negative bacteria 36 ) with either the thrombospondin (TS)1 domain of C6 37 or by its unfurled membrane interacting -hairpin 16 , both of which expose an interface rich in positive charge. Such lipid headgroup dependence also emerges from studies on MAC binding 29,30 and complement activation on model membranes 38 . The observed membrane binding of C5b6 is consistent with recent work on bacteria 9 , reporting that the downstream efficiency of MAC formation is greatly enhanced when C5b6 is actively formed by C5 convertases bound to the bacterial surface.…”

“…To rule out artefacts due to the inherent invasiveness of the AFM measurement, we also performed binding assays by quartz crystal microbalance with dissipation monitoring (QCM-D), which correlate with our AFM data. Briefly, we prepared supported lipid bilayers on a silicon dioxide QCM-D sensor, with which we could detect membrane binding of complement proteins 29,30 (Fig. 2b, SI Fig 5).…”

Immobilization of arrestin-3 on different biosensor platforms for evaluating GPCR binding

“…For all SLB platforms, the final QCM-D measurements corresponded to DF and DD values of À24.04 AE 2.17 Hz and o1 Â 10 À6 , respectively, which agree well with literature values for SLB systems. 34,39,40 In contrast to QCM-D and SPR sensors, BLI sensors are provided with polymer coatings that include Ni-NTA for histidine binding and thus, were not further modified.…”

Materials Nanoarchitectonics for Mechanical Tools in Chemical and Biological Sensing

“…Reprinted with permission from Ref. [74].C opyright 2015 American Chemical Society. demonstrated by using an MSS system with four channels that were coated with poly(vinyl pyrrolidone) (PVP), poly(allylamine hydrochloride) (PAH), poly(methyl methacrylate) (PMMA), and a PVP/PMMA hybrid (1:1 by weight).…”

“…Another advantage of the SLB platform is the possibility to evaluate drug inhibitors that prevent MAC formation and thus mitigate complement activation. Compared with conventional measurement approaches, such as red blood cell assays, the QCM‐D technique has competitive advantages in characterizing the mechanism of action of drug candidates, and it was determined that two MAC inhibitors, vitronectin and clusterin, differentially interfere with MAC assembly at lipid membrane interfaces . For example, vitronectin inhibits complement protein adsorption by binding directly to particular complement proteins and also by competitive adsorption onto the lipid bilayer (Figure ).…”