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Supported Lipid Bilayer Platform to Test Inhibitors of the Membrane Attack Complex: Insights into Biomacromolecular Assembly and Regulation Saziye Yorulmaz, Joshua A Jackman, Walter Hunziker, and Nam-Joon Cho Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b01060 • Publication Date (Web): 07 Oct 2015 Downloaded from http://pubs.acs.org on October 8, 2015

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Biomacromolecules

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Supported Lipid Bilayer Platform to Test Inhibitors

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of the Membrane Attack Complex: Insights into

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Biomacromolecular Assembly and Regulation

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Saziye Yorulmaz†,‡,§, Joshua A. Jackman†,‡,Walter Hunziker§,∥,⊥, Nam-Joon Cho*,†,‡,# †

School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue 639798, Singapore ‡ Centre for Biomimetic Sensor Science, Nanyang Technological University, 50 Nanyang Drive 637553, Singapore § Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Singapore ∥Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore 117599, Singapore ⊥Singapore Eye Research Institute, Singapore # School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive 637459, Singapore *Corresponding author: Nam-Joon Cho E-mail: [email protected] KEYWORDS

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biomacromolecular assembly, biomimetic, supported lipid bilayer, quartz crystal microbalance,

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complement activation, membrane attack complex

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1 ACS Paragon Plus Environment

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ABSTRACT

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Complement activation plays an important role in innate immune defense by triggering

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formation of the membrane attack complex (MAC), which is a biomacromolecular assembly that

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exhibits membrane-lytic activity against foreign invaders, including various pathogens and

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biomaterials. Understanding the details of MAC structure and function has been the subject of

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extensive work involving bulk liposome and erythrocyte assays. However, it is difficult to

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characterize the mechanism of action of MAC inhibitor drug candidates using the conventional

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assays. To address this issue, we employ a biomimetic supported lipid bilayer platform in order

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to investigate how two MAC inhibitors, vitronectin and clusterin, interfere with MAC assembly

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in a sequential addition format, as monitored by the quartz crystal microbalance-dissipation

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(QCM-D) technique. Two experimental strategies based on modular assembly were selected,

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precincubation of inhibitor and C5b-7 complex before addition to the lipid bilayer or initial

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addition of inhibitor followed by the C5b-7 complex. The findings indicate that vitronectin

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inhibits membrane association of C5b-7 via a direct interaction with C5b-7 and via competitive

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membrane association onto the supported lipid bilayer On the other hand, clusterin directly

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interacts with C5b-7 such that C5b-7 is still able to bind to the lipid bilayer and clusterin affects

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the subsequent binding of other complement proteins involved in the MAC assembly. Taken

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together, the findings in this study outline a biomimetic approach based on supported lipid

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bilayers in order to explore the interactions between complement proteins and inhibitors, thereby

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offering insight into MAC assembly and regulation.

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Biomacromolecules

INTRODUCTION

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The complement system is a key part of the innate immune system, which is involved in

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eliminating foreign invaders such as microbes and particulates1-4. Activation of the complement

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system, termed complement activation, consists of a proteolytic cascade involving more than 30

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proteins that form macromolecular complexes in plasma and on membrane surfaces, leading to

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opsonization, anaphylatoxin production, and cytolysis via formation of the membrane attack

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complex (MAC)2, 5. Fundamentally, the mechanisms of complement activation and downstream

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events involve molecular recognition6, with surface features of various pathogens and

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biomaterials inducing activation through either the classical, lectin, or alternative pathway7, 8.

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Recently, a number of studies9,

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activation caused by biomaterials is related to surface characteristics such as the degree of

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hydrophobicity, surface charge density, and the presence or absence of specific functional

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groups11-16. Thus far, attention has been placed on the relationship between complement

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activation and biomaterial properties, with less emphasis on understanding how downstream

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processes such as MAC formation are affected.

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have demonstrated that in vitro and in vivo complement

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The multi-protein MAC induces membrane lysis of target surfaces upon sequential association of

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MAC components that include C5b, C6, C7, C8 and C9 proteins17-19. Initially, C5b interacts with

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C6 and C7 in solution, leading to the formation of a stable C5b-7 complex which can associate

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with target membranes via a hydrophobic domain in C7. Then, C8 and C9 proteins interact with

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the membrane-associated C5b-7 complex in a sequential manner in order to assemble the MAC,

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which leads to formation of a transmembrane pore that causes membrane lysis20-22. Due to the


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potent nature of the functional MAC structure, human cells possess natural inhibitors in the form

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of soluble and membrane-bound proteins which prevent self-destruction23,

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inhibitors such as soluble vitronectin (S-protein), clusterin, and membrane-bound CD59

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selectively block cytolytic MAC assembly25-29. Specifically, vitronectin and clusterin are

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proposed to bind to the C5b-7 precomplex, preventing its association on the cell membrane

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which is a necessary step for MAC assembly30,

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work are still being unraveled and have been the subject of significant experimental efforts.

31

24

. Complement

. The details of how complement inhibitors

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To date, investigation of MAC assembly and its inhibition has focused on human erythrocyte and

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synthetic liposome studies in bulk solution. Hemolysis assays are commonly employed and

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provide a direct readout that is based on membrane lysis. On the other hand, in order to

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characterize formation of the MAC assembly itself, electron microscopy has been employed to

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examine the morphology of MAC assemblies formed by either sequential in situ addition of

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individual MAC components to lipid vesicle solutions or in vitro incubation of erythrocytes in

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human serum20, 32. In addition, light scattering intensity measurements have followed real-time

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MAC assembly by tracking the interaction between MAC components in the presence or absence

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of lipid vesicles33, 34. Furthermore, human, rabbit, or sheep erythrocytes have been incubated in

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human serum followed by detection of membrane-bound and soluble C5b-9 complexes by

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immunoblotting27, 35. Collectively, the existing measurement approaches primarily support direct

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assessment of MAC assembly structure and functional activity. However, the existing

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measurement platforms are difficult to utilize in order to determine the mechanism of action of

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complement inhibitors, lending motivation to the development of new measurement platforms


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Biomacromolecules

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that address this issue, particularly in the context of blocking membrane association of MAC

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components36-38.

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Towards this goal, we recently introduced a supported lipid bilayer (SLB) platform that offers

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label-free investigation of MAC assembly through sequential addition of purified components

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including the C5b-7, C8 and C9 complement proteins. Taking advantage of the compositionally

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flexible solvent-assisted lipid bilayer (SALB) fabrication method, the lipid composition of the

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SLB platform is tunable31, 32 and it was identified that the MAC assembly preferentially forms on

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negatively charged lipid bilayers, in part due to the electrostatic-dependent membrane

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association of the C5b-7 complex39. A key advantage of the SLB platform is its compatibility

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with label-free, surface-sensitive measurement techniques such as the quartz crystal

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microbalance-dissipation (QCM-D) technique which was utilized in the aforementioned study39

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and other biomacromolecular studies40-42. QCM-D measurements can monitor interfacial

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processes including complement protein adsorption and corresponding structural transformations

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involved in MAC assembly by tracking changes in the resonance frequency and energy

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dissipation of a lipid bilayer-coated, oscillating quartz crystal, which are in turn proportional to

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the mass and viscoelastic properties of the adsorbate, respectively43, 44.

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Expanding on the development of an SLB platform to reconstitute the MAC assembly, the goal

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of the present study is to investigate the quantitative stoichiometry of the MAC assembly and to

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determine how two known complement inhibitors, vitronectin and clusterin, function as MAC

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antagonists in the context of lipid membranes. Collectively, our findings support that the SLB

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platform in conjunction with QCM-D monitoring offers a promising approach to characterize the


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mechanism of action of MAC inhibitors, thereby opening the door to the classification of

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existing and investigational drug candidates in this class.

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EXPERIMENTAL SECTION

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Protein Reagents.

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Purified human C5b-6, C7, C8, and C9 native proteins were purchased from Complement Tech

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(Tyler, Texas, USA). The C proteins were stored at -80oC. Human recombinant clusterin (27

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kDa) and vitronectin (52 kDa) proteins were obtained from Mybiosource (San Diego, California,

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USA), and stored at -20oC. Recombinant vitronectin protein was received in lyophilized powder

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form and reconstituted at a stock concentration of 1 mg/mL in water containing 0.1 wt% bovine

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serum albumin (BSA). Recombinant clusterin protein was received in phosphate-buffered saline

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(PBS) [pH 7.4] containing 50% glycerol. Immediately before experiment, all proteins were

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diluted in 10 mM Tris buffer [pH 7.5] with 150 mM NaCl to the desired protein concentration.

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Quartz Crystal Microbalance-Dissipation.

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A Q-Sense E4 instrument (Q-sense AB, Gothenburg, Sweden) was utilized in order to monitor

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lipid and protein adsorption processes by tracking the changes in resonance frequency and

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energy dissipation of a silicon oxide-coated quartz crystal (model no. QSX303) as functions of

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time. The QCM-D measurement data were collected at the 3rd, 5th, 7th, 9th, and 11th odd

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overtones, and the reported QCM-D data were all obtained at the 5th overtone and normalized


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Biomacromolecules

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accordingly (Δfn=5/5). Immediately before experiment, the QCM-D sensor substrates were

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sequentially rinsed with 2% SDS, water, and ethanol followed by drying with nitrogen gas. The

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substrates were then subjected to oxygen plasma treatment (Harrick Plasma, Ithaca, NY) at the

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maximum radiofrequency (RF) power. All QCM-D measurements were conducted under

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continuous flow conditions, with the flow rate defined as 100µL/min for all steps in the bilayer

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formation process and 41.8µL/min for protein addition as controlled by a Reglo Digital

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peristaltic pump (Ismatec, Glattbrugg, Switzerland). The temperature of the flow cell was fixed

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at 24.00 ± 0.5 °C. In order to estimate the molecular ratio of adsorbed proteins, the Sauerbrey

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model was applied in order to convert frequency shifts into mass density values as follows:

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𝛥𝑚 = −𝐶

𝛥𝑓𝑛 𝑛

,

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where Δm is the adsorbate mass, C is the proportionality constant (17.7 ng/cm2), Δf is the

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frequency shift, and n is the odd overtone number of the resonance frequency45.

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Supported Lipid Bilayer Formation.

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The solvent-assisted lipid bilayer (SALB) formation method was used in order to prepare

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supported lipid bilayers. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-

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oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) were purchased in

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lyophilized powder form from Avanti Polar Lipids (Alabaster, AL). Before experiment, DOPC

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lipid powder was dissolved in isopropanol at a stock concentration of 10 mg/mL. POPG lipid

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powder was dissolved in ethanol at a stock concentration of 5 mg/mL and heated to around 40°C

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in order to promote solubilization. Then, a 70%/30% molar fraction of DOPC and POPG lipids

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was prepared by mixing lipids and diluted in isopropanol to a final concentration of 0.5 mg/mL.


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As part of the SALB experimental protocol, a measurement baseline was first recorded in

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aqueous buffer solution (10 mM Tris [pH 7.5] with 150 mM NaCl) (Step 1), followed by

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exchange with isopropanol solution (Step 2). Then, 0.5 mg/mL lipid in isopropanol was

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deposited onto the substrate (Step 3), and incubated for approximately 10 min before a solvent-

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exchange step was performed as aqueous buffer solution was reintroduced leading to bilayer

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formation (Step 4). After bilayer formation, 0.1 mg/mLl BSA was added (Step 5) in order to

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verify the completeness of bilayer formation.

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Membrane Attack Complex Assay.

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The C5b-6, C7, C8 and C9 proteins were used in a sequential addition assay format in order to

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establish the membrane attack complex on the supported lipid bilayer platform, as monitored by

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the QCM-D technique. Before experiment, protein aliquots were diluted with aqueous buffer

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solution (10 mM Tris, 150 mM NaCl [pH 7.5]). The C5b-5 and C6 proteins were pre-complexed

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in order to form a conjugate for membrane association. The C5b-7 complex was formed by

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mixing 20 nM C5b-6 and 50 nM C7 for a 30 min period of incubation at room temperature.

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Then, the C5b-7 precomplex was added to the supported lipid bilayer, followed by sequential

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addition of 30 nM C8 protein and then 200 nM C9 protein.

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MAC Inhibitor Studies.

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The inhibitory effects of recombinant clusterin and vitronectin proteins on MAC assembly were

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evaluated by using the supported lipid bilayer platform. To investigate the mechanism of


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Biomacromolecules

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inhibition, the inhibitor proteins were individually studied and either mixed with C5b-7

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precomplex or added beforehand to the supported lipid bilayer platform (Scheme 1). In the first

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experimental format, varying concentrations of clusterin or vitronectin between 1 and 200 nM

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were mixed with 20 nM C5b-7 complex and incubated at room temperature for 30 min before

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experiment. Then, the solution containing C5b-7 complex and inhibitor was added to the bilayer

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before sequential addition of C8 and C9 proteins. Alternatively, varying concentrations of

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clusterin or vitronectin between 1 and 200 nM were added to the bilayer followed by sequential

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addition of C5b-7, C8, and C9 proteins. As mentioned above, stock clusterin was supplied in a

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50% glycerol solution. It is important to take into account the specific glycerol fraction because

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glycerol affects the solution viscosity and QCM-D measurements are sensitive to changes in the

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viscosity of the bulk solution. For this reason, we carefully performed measurements in order to

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verify that glycerol has negligible effects on the supported lipid bilayer (Figure S1). In the

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clusterin experiments, the supported lipid bilayer was initially formed in pure aqueous buffer

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solution and then the solution was exchanged with aqueous buffer solution which contained a

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glycerol fraction equivalent to that of the clusterin solution prepared for the given experiment.

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Upon exchange with the glycerol-water mixture, minor frequency and dissipation shifts were

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observed, as expected due to the change in solution viscosity. After clusterin and C5b-7 addition

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was performed via either strategy, exchange with aqueous buffer solution was completed and

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additional steps in the MAC formation process (C8 and C9 protein addition) were conducted in

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pure aqueous buffer solution.


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Scheme 1. Experimental Strategies to Test Inhibitors of MAC Assembly. Two strategies

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were employed. In the first strategy, (i) the inhibitor and C5b-7 complex were mixed in solution

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and then (ii) the mixture was added to the SLB platform, followed by sequential addition of (iii)

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C8 and (iv) C9 proteins. In the second case, (i) the inhibitor was first added to the SLB platform

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followed by sequential addition of (ii) C5b-7, (iii) C8 and (iv) C9 proteins to the SLB platform. 10 ACS Paragon Plus Environment

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Biomacromolecules

RESULTS AND DISCUSSION

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Design of MAC Assay Platform

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In order to test the effect of inhibitors on MAC assembly, we first established a supported lipid

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bilayer platform on silicon oxide. The bilayer was fabricated by using the SALB method39, 46, 47,

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which involves the deposition of lipids dissolved in alcohol onto the solid support followed by

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solvent-exchange with aqueous buffer solution to promote bilayer formation. The selected lipid

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composition was 70 mol% DOPC and 30 mol% POPG in order to prepare negatively charged

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supported lipid bilayers which support MAC protein adsorption39. The QCM-D technique allows

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label-free measurement of the mass and viscoelastic properties of the adsorbed material, enabling

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detailed characterization of the fabricated lipid bilayer and each step of protein addition. After

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forming the lipid bilayer, the final frequency and dissipation shifts are -25.0 ± 1.4 Hz and 0.6 ±

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0.3 x 10-6, respectively, and the values are in agreement with past reported values39, 46, 47 (Figure

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S2). An additional step of BSA protein addition to bare and bilayer-coated substrates indicated

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that the bilayers are >95% complete and passivated remaining parts of the substrate in order to

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specifically detect membrane association of MAC proteins to the lipid bilayer (Figure S3).

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Thereafter, as part of the MAC assay described in Figure 1A, the change in frequency shift was

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recorded as a function of time upon addition of protein components of the membrane attack

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complex, with C5b-7, C8 and C9 proteins added in sequential format (Figure 1B).

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Each stage of protein addition (indicated by the respective arrows) led to a negative frequency

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shift. Because the frequency shift is negatively proportional to the adsorbed mass, the frequency


Biomacromolecules

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shifts indicate that the proteins attach to the lipid bilayer and the adsorbed mass of each protein

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can be quantified (Figure 1C). By taking into account the molecular weights of each protein, the

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molar ratio of bound proteins can be determined. Based on this approach, it

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Figure 1. QCM-D Monitoring of MAC Assembly on the SLB Platform. (A) Schematic

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illustration of MAC formation via sequential addition format. Changes in (B) frequency and (C)

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Sauerbrey mass were monitored as functions of time after sequential addition of C5b-7 complex,

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C8 protein, and C9 protein onto the 70:30 mol% DOPC:POPG SLB platform. The SLB on

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silicon oxide was initially formed by the SALB method, and then the measurement time was

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normalized. At t = 0 min, the MAC assay was initiated and the C5b-7 complex was added at t =

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20 min. Then, C8 protein was added at t = 80 min and C9 protein at t = 150 min. The adsorbed

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mass was calculated by the Sauerbrey relationship and the mass sensitivity constant for a 1Hz

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frequency shift is 17.7 ng/cm2.


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was determined that approximately 1.25 C8 and 3.60 C9 proteins were attached to the lipid

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bilayer per 1 C5b-7 protein. The calculated C5b-7:C8 molar ratio is in good agreement with

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literature reports that indicate a 1:1 stoichiometry33. From these calculations, we also determined

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that the measured stoichiometry of the C5b-8 intermediate complex to C9 protein is 3.4, which

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agrees well with the literature range between 3 and 1627,

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indicate that the platform offers a quantifiable, label-free assessment of bound protein mass,

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leading us to investigate how the two inhibitors each affect MAC assembly.

33, 48, 49

. Taken together, the results

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Effect of Vitronectin on MAC Assembly

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Using the supported lipid bilayer platform, we first investigated the effect of vitronectin on

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membrane association of the C5b-7 complex. The C5b-7 complex and vitronectin were mixed

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before experiment, and then added to the lipid bilayer platform (Figure 2A). With increasing

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vitronectin concentration, there was a general decrease in the frequency shift associated with

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protein adsorption (Figure 2B). In order to investigate details of the vitronectin concentration-

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dependent effects, the maximum frequency shifts are presented as a function of vitronectin

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concentration in Figure 2C. Interestingly, at lower vitronectin concentrations (5 nM and below),

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the frequency shifts were lower than the value obtained from the association of C5b-7 complex

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in the absence of inhibitor. These results support that some of the C5b-7 complex interacted with

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vitronectin in the solution and loses the ability to associate on the bilayer at lower vitronectin

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concentration. That is, when vitronectin directly interacts with C5b-7 in solution, the resulting

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vitronectin-C5b-7 complex is prevented from subsequently binding to the lipid bilayer. By

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contrast, at higher vitronectin concentrations, there is a clear increase in the total adsorbed


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Figure 2. Influence of Vitronectin on Membrane Association of the C5b-7 Complex. (A)

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Schematic illustration shows addition of the C5b-7 complex which was preincubated with

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vitronectin. Panel (B) presents the QCM-D frequency shift associated with C5b-7 + vitronectin

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addition to the SLB platform as a function of vitronectin concentration. The C5b-7 complex

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concentration was fixed at 20 nM. The proteins were added at t = 20 min. Panel (C) shows

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absolute value of the maximum QCM-D frequency shift due to the amount of total adsorbed

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protein as a function of vitronectin concentration based on the data presented in Panel (B). Each

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data point is the average + standard deviation of three independent (n = 3) measurements. (D-E)

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Corresponding schematic illustration and QCM-D measurement data for the addition of

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vitronectin to the SLB platform, followed by addition of C5b-7 complex to the same platform.

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Panel (F) shows absolute values of the maximum QCM-D frequency shifts due to the amounts of

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adsorbed vitronectin and C5b-7 complex as a function of vitronectin concentration based on the

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data presented in Panel (E). Each data point is the average + standard deviation of three

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independent (n = 3) measurements. 14 ACS Paragon Plus Environment

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Biomacromolecules

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protein mass. In particular, at 100 nM and higher vitronectin concentrations, there are large

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frequency shifts which suggest that vitronectin was the dominant adsorbed species because the

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C5b-7 protein concentration was fixed at 20 nM.

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In order to clarify the effect of vitronectin on C5b-7 adsorption, a sequential addition protocol

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was followed in parallel experiments across the same range of inhibitor concentrations (Figure

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2D). Figure 2E presents the individual frequency shifts associated with membrane association of

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vitronectin followed by C5b-7 protein. Again, with increasing vitronectin concentration, there

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was an increase in vitronectin uptake. On the other hand, increasing concentrations of vitronectin

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inhibited subsequent membrane association of C5b-7. As presented in Figure 2F, the frequency

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shifts were proportional to the vitronectin concentration ranging up to -19 Hz at 200 nM

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concentration. Likewise, prior membrane association of vitronectin suppressed membrane

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association of C5b-7, with negligible C5b-7 addition at high vitronectin concentrations.

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Particularly strong inhibition was observed at vitronectin concentrations above 50nM. Taken

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together, the data obtained from both experimental strategies support that vitronectin inhibits

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membrane association of C5b-7 via direct interaction with C5b-7 which prevents binding to the

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lipid bilayer and via competitive membrane association onto the supported lipid bilayer which

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restricts the number of available adsorption sites.

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Following addition of the i) premixed C5b-7 and vitronectin or ii) vitronectin followed by C5b-7,

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there was next sequential addition of C8 and C9 proteins in order to investigate the effect of

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surface-bound vitronectin. In Figure 3, the frequency shifts associated with C8 and C9 protein


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addition are presented as a function of vitronectin concentration. Independent of the route by

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which the supported lipid bilayer was treated with vitronectin, subsequent membrane association

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Figure 3. Effect of Vitronectin on the Sequential Addition of C8 and C9 Proteins to the

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SLB Platform. (A) Schematic illustration of sequential addition of C8 protein to the SLB

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platform after vitronectin and C5b-7 addition. (B) QCM-D maximum frequency shift associated

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with C8 protein addition as a function of vitronectin concentration for each strategy. Each data

321

point is the average + standard deviation of three independent (n = 3) measurements. (C-D)

322

Corresponding schematic illustration and QCM-D measurement data for C9 protein addition as a

323

function of vitronectin concentration. 16 ACS Paragon Plus Environment

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of C8 and C9 proteins decreased with increasing vitronectin concentration. There is a nearly

325

linear trend in inhibition between 5 and 100 nM vitronectin, with minimal inhibition below this

326

concentration range. Collectively, the results are consistent with vitronectin blocking membrane

327

association of C5b-7, in turn preventing membrane association of C8 and C9 proteins as well.

328 329

Effect of Clusterin on MAC Assembly

330 331

In addition to vitronectin, we also studied the inhibitory effect of clusterin on membrane

332

association of the C5b-7 complex. Initially, clusterin and C5b-7 complex were premixed and

333

incubated for 30 min at room temperature (Figure 4A). Figure 4B presents the QCM-D

334

frequency shifts for the addition of premixed C5b-7 and clusterin to the SLB platform. With

335 336

Figure 4. Influence of Clusterin on Membrane Association of the C5b-7 Complex. (A)

337

Schematic illustration shows addition of the C5b-7 complex which was preincubated with

338

clusterin. Panel (B) presents the QCM-D frequency shift associated with C5b-7 + clusterin 17 ACS Paragon Plus Environment

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addition to the SLB platform as a function of vitronectin concentration. The C5b-7 complex

340

concentration was fixed at 20 nM. The proteins were added at t = 20 min. Panel (C) shows

341

absolute value of the maximum QCM-D frequency shift due to the amount of total adsorbed

342

protein as a function of clusterin concentration based on the data presented in Panel (B). Each

343

data point is the average + standard deviation of three independent (n = 3) measurements. (D-E)

344

Corresponding schematic illustration and QCM-D measurement data for the addition of clusterin

345

to the SLB platform, followed by addition of C5b-7 complex to the same platform. Panel (F)

346

shows absolute values of the maximum QCM-D frequency shifts due to the amounts of adsorbed

347

clusterin and C5b-7 complex as a function of clusterin concentration based on the data presented

348

in Panel (E). Each data point is the average + standard deviation of three independent (n = 3)

349

measurements.

350 351

increasing clusterin concentration, there was a trend towards increased frequency shifts as

352

reflected in Figure 4C. Low clusterin concentrations had negligible effect on protein uptake,

353

while there was a linear correspondence between clusterin concentration and protein uptake at 10

354

nM clusterin and above. The data indicate that clusterin attached to the lipid bilayer together with

355

the C5b-7 complex, clarifying a previous report50 which suggested that clusterin prevents

356

attachment of C5b-7 complex to the membrane. In parallel experiments, clusterin was directly

357

added to the supported lipid bilayer in the absence of the C5b-7 complex (Figure 4D). Even at

358

high clusterin concentrations, there was only minimal attachment to the lipid bilayer (less than 5

359

Hz in all cases) (Figure 4E). In turn, the amount of attached C5b-7 on the lipid bilayer surface

360

did not depend on the bulk clusterin concentration (Figure 4F).


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In order to further investigate the effect of clusterin on membrane association of C8 and C9

362

proteins, C8 and C9 proteins were sequentially added after initial addition of premixed clusterin-

363

C5b-7 or after sequential addition of clusterin and then the C5b-7 complex. Changes in

364

frequency shifts associated with C8 protein addition are presented as a concentration of clusterin

365

(Figures 5A-B). For both experimental strategies, the changes in frequency shift were largely

366 367

Figure 5. Effect of clusterin on the sequential addition of C8 and C9 proteins to the SLB

368

platform. (A) Schematic illustration of sequential addition of C8 protein to the SLB platform

369

after clusterin and C5b-7 addition. (B) QCM-D maximum frequency shift associated with C8


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protein addition as a function of clusterin concentration for each strategy. Each data point is the

371

average + standard deviation of three independent (n = 3) measurements. (C-D) Corresponding

372

schematic illustration and QCM-D measurement data for C9 protein addition as a function of

373

clusterin concentration.

374

independent of clusterin concentration at low and moderate inhibitor concentrations whereas

375

there was some inhibition of C8 protein attachment at high concentrations (20 nM clusterin and

376

above). This moderate inhibitory effect of clusterin on C8 protein attachment is consistent with

377

previous dye release experiments28. After C8 addition, C9 protein was next added and the

378

changes in frequency shifts are presented as a function of clusterin concentration (Figures 5C-

379

D). Interestingly, the amount of bound C9 increased with increasing clusterin concentration,

380

which is also consistent with the previous finding25 that clusterin is known to bind to multiple

381

sites on C9.

382

Comparison of Inhibition by Clusterin versus Vitronectin

383 384

Herein, we have compared the inhibitory activity of recombinant vitronectin and clusterin

385

proteins on MAC assembly. The interaction of vitronectin and clusterin with supported lipid

386

bilayers was determined by using the QCM-D technique. Figure 6A presents the changes in

387

attached protein mass density upon addition of vitronectin and clusterin as a function of protein

388

concentrations. The adsorbed mass of vitronectin proteins increased when the concentration of

389

vitronectin increased ranging from 12 to 344 ng/cm2, and occurs due to electrostatic attraction

390

because vitronectin has a positively charged domain31 that prefers membrane association onto

391

negatively charged lipid bilayers. However, when the C5b-7 complex was incubated with

392

vitronectin, vitronectin blocked subsequent membrane association of the C5b-7 complex at lower 20 ACS Paragon Plus Environment

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concentrations which results in less adsorbed protein, as reflected in Figure 6B. The higher

394

concentration of vitronectin induced an increase in total protein attachment due to adsorption of

395

both C5b-7 complex and vitronectin. These findings are consistent with past reports31,

396

vitronectin inhibits MAC formation through occupation of the membrane binding site of the

397

C5b-7 complex and extends this knowledge to directly show that vitronectin has a high

398

propensity to attach to negatively charged lipid bilayers.

399 400

Figure 6. Comparison of Inhibition by Vitronectin and Clusterin on Membrane Association

401

of the C5b-7 Complex. (A) Changes in adsorbed mass of vitronectin and clusterin upon addition


51

that

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402

to the SLB platform as a function of inhibitor concentration. (B) Changes in adsorbed mass due

403

to inhibitor + C5b-7 complex upon joint addition to the SLB platform as a function of inhibitor

404

concentration. Each data point is the average + standard deviation of three independent (n = 3)

405

measurements.

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406 407

In the case of clusterin, there was only minor adsorption of the inhibitor to the SLB platform in

408

all cases, with mass shifts always below 50 ng/cm2 (Figure 6A). Although clusterin did not

409

directly interact with the SLB platform, the preincubation of clusterin with the C5b-7 complex

410

induced an appreciable change in the attached mass of total protein which depended on the

411

clusterin concentration as depicted in Figure 6B. Based on these results, we conclude that

412

clusterin attached to the C5b-7 complex but did not block the membrane association site of the

413

C5b-7 complex. Collectively, the QCM-D measurement results obtained on the SLB platform

414

demonstrate that vitronectin and clusterin differentially interfere with the C5b-7 complex as part

415

of their roles as complement inhibitors. Looking forward, there is excellent potential to utilize

416

the SLB platform with additional sensing modalities such as combined QCM-D and electrical

417

impedance spectroscopy measurements52 in order to investigate how complement inhibitors

418

interfere with other processes involved in MAC assembly such as pore formation.

419 420

CONCLUSION

421 422

In this study, we have investigated the association of MAC components onto the SLB platform in

423

the presence and absence of known MAC inhibitors. First, we measured the quantitative

424

stoichiometry of adsorbed MAC components on negatively charged SLBs. Compared to existing


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425

attempts to determine stoichiometry from light scattering and electron microscopy, our QCM-D

426

measurement approach yields high precision data based on label-free mass-sensitive detection.

427

Extending these capabilities, the specific inhibitory mechanisms of vitronectin and clusterin

428

towards the MAC assembly were identified using a combination of two experimental strategies

429

which focused on the direct protein-protein interaction with the C5b-7 complex and competitive

430

membrane association of the inhibitor to the SLB. The findings support that vitronectin blocked

431

the membrane association of the C5b-7 complex to the SLB platform at low inhibitor

432

concentrations through a direct interaction whereas vitronectin at higher concentrations

433

principally acted via competitive attachment to the SLB platform. By contrast, clusterin directly

434

interacted with the C5b-7 complex and the two proteins adsorbed together onto the SLB. Taken

435

together, the results highlight the capabilities of the SLB platform to test inhibitors of the MAC

436

assembly, generating critical mechanistic information to guide drug discovery and development

437

with broad applicability to additional classes of inhibitors including small molecules, antibodies

438

and peptides.

439 440

ASSOCIATED CONTENT

441 442

The Supporting Information is available free of charge on the ACS Publications website at DOI:

443

QCM-D characterization of supported lipid bilayer formation and analysis of the lipid

444

bilayer homogeneity.

445 446 447


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448

AUTHOR INFORMATION

449 450

Corresponding Author

451

*E-mail: [email protected]

452 453

Author Contributions

454

The manuscript was written through contributions of all authors. All authors have given approval

455

to the final version of the manuscript.

456 457

Notes

458

The authors declare no competing financial interest.

459 460

ACKNOWLEGEMENTS

461 462

This work was supported by the National Research Foundation grant NRF-NRFF2011-01, the

463

National Medical Research Council grant NMRC/CBRG/0005/2012, and the A*STAR-NHG-

464

NTU Skin Research Grant (SRG/14028) to N.J.C

465 466 467 468 469 470


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