Subscriber access provided by NEW YORK MED COLL
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
1
Supported Lipid Bilayer Platform to Test Inhibitors
2
of the Membrane Attack Complex: Insights into
3
Biomacromolecular Assembly and Regulation
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
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
26
biomacromolecular assembly, biomimetic, supported lipid bilayer, quartz crystal microbalance,
27
complement activation, membrane attack complex
28 29 30 31
1 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
32
ABSTRACT
33 34
Complement activation plays an important role in innate immune defense by triggering
35
formation of the membrane attack complex (MAC), which is a biomacromolecular assembly that
36
exhibits membrane-lytic activity against foreign invaders, including various pathogens and
37
biomaterials. Understanding the details of MAC structure and function has been the subject of
38
extensive work involving bulk liposome and erythrocyte assays. However, it is difficult to
39
characterize the mechanism of action of MAC inhibitor drug candidates using the conventional
40
assays. To address this issue, we employ a biomimetic supported lipid bilayer platform in order
41
to investigate how two MAC inhibitors, vitronectin and clusterin, interfere with MAC assembly
42
in a sequential addition format, as monitored by the quartz crystal microbalance-dissipation
43
(QCM-D) technique. Two experimental strategies based on modular assembly were selected,
44
precincubation of inhibitor and C5b-7 complex before addition to the lipid bilayer or initial
45
addition of inhibitor followed by the C5b-7 complex. The findings indicate that vitronectin
46
inhibits membrane association of C5b-7 via a direct interaction with C5b-7 and via competitive
47
membrane association onto the supported lipid bilayer On the other hand, clusterin directly
48
interacts with C5b-7 such that C5b-7 is still able to bind to the lipid bilayer and clusterin affects
49
the subsequent binding of other complement proteins involved in the MAC assembly. Taken
50
together, the findings in this study outline a biomimetic approach based on supported lipid
51
bilayers in order to explore the interactions between complement proteins and inhibitors, thereby
52
offering insight into MAC assembly and regulation.
53 54
2 ACS Paragon Plus Environment
Page 2 of 29
Page 3 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
55
Biomacromolecules
INTRODUCTION
56 57
The complement system is a key part of the innate immune system, which is involved in
58
eliminating foreign invaders such as microbes and particulates1-4. Activation of the complement
59
system, termed complement activation, consists of a proteolytic cascade involving more than 30
60
proteins that form macromolecular complexes in plasma and on membrane surfaces, leading to
61
opsonization, anaphylatoxin production, and cytolysis via formation of the membrane attack
62
complex (MAC)2, 5. Fundamentally, the mechanisms of complement activation and downstream
63
events involve molecular recognition6, with surface features of various pathogens and
64
biomaterials inducing activation through either the classical, lectin, or alternative pathway7, 8.
65
Recently, a number of studies9,
66
activation caused by biomaterials is related to surface characteristics such as the degree of
67
hydrophobicity, surface charge density, and the presence or absence of specific functional
68
groups11-16. Thus far, attention has been placed on the relationship between complement
69
activation and biomaterial properties, with less emphasis on understanding how downstream
70
processes such as MAC formation are affected.
10
have demonstrated that in vitro and in vivo complement
71 72
The multi-protein MAC induces membrane lysis of target surfaces upon sequential association of
73
MAC components that include C5b, C6, C7, C8 and C9 proteins17-19. Initially, C5b interacts with
74
C6 and C7 in solution, leading to the formation of a stable C5b-7 complex which can associate
75
with target membranes via a hydrophobic domain in C7. Then, C8 and C9 proteins interact with
76
the membrane-associated C5b-7 complex in a sequential manner in order to assemble the MAC,
77
which leads to formation of a transmembrane pore that causes membrane lysis20-22. Due to the
3 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 29
78
potent nature of the functional MAC structure, human cells possess natural inhibitors in the form
79
of soluble and membrane-bound proteins which prevent self-destruction23,
80
inhibitors such as soluble vitronectin (S-protein), clusterin, and membrane-bound CD59
81
selectively block cytolytic MAC assembly25-29. Specifically, vitronectin and clusterin are
82
proposed to bind to the C5b-7 precomplex, preventing its association on the cell membrane
83
which is a necessary step for MAC assembly30,
84
work are still being unraveled and have been the subject of significant experimental efforts.
31
24
. Complement
. The details of how complement inhibitors
85 86
To date, investigation of MAC assembly and its inhibition has focused on human erythrocyte and
87
synthetic liposome studies in bulk solution. Hemolysis assays are commonly employed and
88
provide a direct readout that is based on membrane lysis. On the other hand, in order to
89
characterize formation of the MAC assembly itself, electron microscopy has been employed to
90
examine the morphology of MAC assemblies formed by either sequential in situ addition of
91
individual MAC components to lipid vesicle solutions or in vitro incubation of erythrocytes in
92
human serum20, 32. In addition, light scattering intensity measurements have followed real-time
93
MAC assembly by tracking the interaction between MAC components in the presence or absence
94
of lipid vesicles33, 34. Furthermore, human, rabbit, or sheep erythrocytes have been incubated in
95
human serum followed by detection of membrane-bound and soluble C5b-9 complexes by
96
immunoblotting27, 35. Collectively, the existing measurement approaches primarily support direct
97
assessment of MAC assembly structure and functional activity. However, the existing
98
measurement platforms are difficult to utilize in order to determine the mechanism of action of
99
complement inhibitors, lending motivation to the development of new measurement platforms
4 ACS Paragon Plus Environment
Page 5 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
100
that address this issue, particularly in the context of blocking membrane association of MAC
101
components36-38.
102 103
Towards this goal, we recently introduced a supported lipid bilayer (SLB) platform that offers
104
label-free investigation of MAC assembly through sequential addition of purified components
105
including the C5b-7, C8 and C9 complement proteins. Taking advantage of the compositionally
106
flexible solvent-assisted lipid bilayer (SALB) fabrication method, the lipid composition of the
107
SLB platform is tunable31, 32 and it was identified that the MAC assembly preferentially forms on
108
negatively charged lipid bilayers, in part due to the electrostatic-dependent membrane
109
association of the C5b-7 complex39. A key advantage of the SLB platform is its compatibility
110
with label-free, surface-sensitive measurement techniques such as the quartz crystal
111
microbalance-dissipation (QCM-D) technique which was utilized in the aforementioned study39
112
and other biomacromolecular studies40-42. QCM-D measurements can monitor interfacial
113
processes including complement protein adsorption and corresponding structural transformations
114
involved in MAC assembly by tracking changes in the resonance frequency and energy
115
dissipation of a lipid bilayer-coated, oscillating quartz crystal, which are in turn proportional to
116
the mass and viscoelastic properties of the adsorbate, respectively43, 44.
117 118
Expanding on the development of an SLB platform to reconstitute the MAC assembly, the goal
119
of the present study is to investigate the quantitative stoichiometry of the MAC assembly and to
120
determine how two known complement inhibitors, vitronectin and clusterin, function as MAC
121
antagonists in the context of lipid membranes. Collectively, our findings support that the SLB
122
platform in conjunction with QCM-D monitoring offers a promising approach to characterize the
5 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
123
mechanism of action of MAC inhibitors, thereby opening the door to the classification of
124
existing and investigational drug candidates in this class.
125 126
EXPERIMENTAL SECTION
127 128
Protein Reagents.
129 130
Purified human C5b-6, C7, C8, and C9 native proteins were purchased from Complement Tech
131
(Tyler, Texas, USA). The C proteins were stored at -80oC. Human recombinant clusterin (27
132
kDa) and vitronectin (52 kDa) proteins were obtained from Mybiosource (San Diego, California,
133
USA), and stored at -20oC. Recombinant vitronectin protein was received in lyophilized powder
134
form and reconstituted at a stock concentration of 1 mg/mL in water containing 0.1 wt% bovine
135
serum albumin (BSA). Recombinant clusterin protein was received in phosphate-buffered saline
136
(PBS) [pH 7.4] containing 50% glycerol. Immediately before experiment, all proteins were
137
diluted in 10 mM Tris buffer [pH 7.5] with 150 mM NaCl to the desired protein concentration.
138 139
Quartz Crystal Microbalance-Dissipation.
140 141
A Q-Sense E4 instrument (Q-sense AB, Gothenburg, Sweden) was utilized in order to monitor
142
lipid and protein adsorption processes by tracking the changes in resonance frequency and
143
energy dissipation of a silicon oxide-coated quartz crystal (model no. QSX303) as functions of
144
time. The QCM-D measurement data were collected at the 3rd, 5th, 7th, 9th, and 11th odd
145
overtones, and the reported QCM-D data were all obtained at the 5th overtone and normalized
6 ACS Paragon Plus Environment
Page 6 of 29
Page 7 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
146
accordingly (Δfn=5/5). Immediately before experiment, the QCM-D sensor substrates were
147
sequentially rinsed with 2% SDS, water, and ethanol followed by drying with nitrogen gas. The
148
substrates were then subjected to oxygen plasma treatment (Harrick Plasma, Ithaca, NY) at the
149
maximum radiofrequency (RF) power. All QCM-D measurements were conducted under
150
continuous flow conditions, with the flow rate defined as 100µL/min for all steps in the bilayer
151
formation process and 41.8µL/min for protein addition as controlled by a Reglo Digital
152
peristaltic pump (Ismatec, Glattbrugg, Switzerland). The temperature of the flow cell was fixed
153
at 24.00 ± 0.5 °C. In order to estimate the molecular ratio of adsorbed proteins, the Sauerbrey
154
model was applied in order to convert frequency shifts into mass density values as follows:
155
𝛥𝑚 = −𝐶
𝛥𝑓𝑛 𝑛
,
156
where Δm is the adsorbate mass, C is the proportionality constant (17.7 ng/cm2), Δf is the
157
frequency shift, and n is the odd overtone number of the resonance frequency45.
158 159
Supported Lipid Bilayer Formation.
160 161
The solvent-assisted lipid bilayer (SALB) formation method was used in order to prepare
162
supported lipid bilayers. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-
163
oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (POPG) were purchased in
164
lyophilized powder form from Avanti Polar Lipids (Alabaster, AL). Before experiment, DOPC
165
lipid powder was dissolved in isopropanol at a stock concentration of 10 mg/mL. POPG lipid
166
powder was dissolved in ethanol at a stock concentration of 5 mg/mL and heated to around 40°C
167
in order to promote solubilization. Then, a 70%/30% molar fraction of DOPC and POPG lipids
168
was prepared by mixing lipids and diluted in isopropanol to a final concentration of 0.5 mg/mL.
7 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
169
As part of the SALB experimental protocol, a measurement baseline was first recorded in
170
aqueous buffer solution (10 mM Tris [pH 7.5] with 150 mM NaCl) (Step 1), followed by
171
exchange with isopropanol solution (Step 2). Then, 0.5 mg/mL lipid in isopropanol was
172
deposited onto the substrate (Step 3), and incubated for approximately 10 min before a solvent-
173
exchange step was performed as aqueous buffer solution was reintroduced leading to bilayer
174
formation (Step 4). After bilayer formation, 0.1 mg/mLl BSA was added (Step 5) in order to
175
verify the completeness of bilayer formation.
176 177
Membrane Attack Complex Assay.
178 179
The C5b-6, C7, C8 and C9 proteins were used in a sequential addition assay format in order to
180
establish the membrane attack complex on the supported lipid bilayer platform, as monitored by
181
the QCM-D technique. Before experiment, protein aliquots were diluted with aqueous buffer
182
solution (10 mM Tris, 150 mM NaCl [pH 7.5]). The C5b-5 and C6 proteins were pre-complexed
183
in order to form a conjugate for membrane association. The C5b-7 complex was formed by
184
mixing 20 nM C5b-6 and 50 nM C7 for a 30 min period of incubation at room temperature.
185
Then, the C5b-7 precomplex was added to the supported lipid bilayer, followed by sequential
186
addition of 30 nM C8 protein and then 200 nM C9 protein.
187 188
MAC Inhibitor Studies.
189 190
The inhibitory effects of recombinant clusterin and vitronectin proteins on MAC assembly were
191
evaluated by using the supported lipid bilayer platform. To investigate the mechanism of
8 ACS Paragon Plus Environment
Page 8 of 29
Page 9 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
192
inhibition, the inhibitor proteins were individually studied and either mixed with C5b-7
193
precomplex or added beforehand to the supported lipid bilayer platform (Scheme 1). In the first
194
experimental format, varying concentrations of clusterin or vitronectin between 1 and 200 nM
195
were mixed with 20 nM C5b-7 complex and incubated at room temperature for 30 min before
196
experiment. Then, the solution containing C5b-7 complex and inhibitor was added to the bilayer
197
before sequential addition of C8 and C9 proteins. Alternatively, varying concentrations of
198
clusterin or vitronectin between 1 and 200 nM were added to the bilayer followed by sequential
199
addition of C5b-7, C8, and C9 proteins. As mentioned above, stock clusterin was supplied in a
200
50% glycerol solution. It is important to take into account the specific glycerol fraction because
201
glycerol affects the solution viscosity and QCM-D measurements are sensitive to changes in the
202
viscosity of the bulk solution. For this reason, we carefully performed measurements in order to
203
verify that glycerol has negligible effects on the supported lipid bilayer (Figure S1). In the
204
clusterin experiments, the supported lipid bilayer was initially formed in pure aqueous buffer
205
solution and then the solution was exchanged with aqueous buffer solution which contained a
206
glycerol fraction equivalent to that of the clusterin solution prepared for the given experiment.
207
Upon exchange with the glycerol-water mixture, minor frequency and dissipation shifts were
208
observed, as expected due to the change in solution viscosity. After clusterin and C5b-7 addition
209
was performed via either strategy, exchange with aqueous buffer solution was completed and
210
additional steps in the MAC formation process (C8 and C9 protein addition) were conducted in
211
pure aqueous buffer solution.
9 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 10 of 29
212 213
Scheme 1. Experimental Strategies to Test Inhibitors of MAC Assembly. Two strategies
214
were employed. In the first strategy, (i) the inhibitor and C5b-7 complex were mixed in solution
215
and then (ii) the mixture was added to the SLB platform, followed by sequential addition of (iii)
216
C8 and (iv) C9 proteins. In the second case, (i) the inhibitor was first added to the SLB platform
217
followed by sequential addition of (ii) C5b-7, (iii) C8 and (iv) C9 proteins to the SLB platform. 10 ACS Paragon Plus Environment
Page 11 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
218
Biomacromolecules
RESULTS AND DISCUSSION
219 220
Design of MAC Assay Platform
221 222
In order to test the effect of inhibitors on MAC assembly, we first established a supported lipid
223
bilayer platform on silicon oxide. The bilayer was fabricated by using the SALB method39, 46, 47,
224
which involves the deposition of lipids dissolved in alcohol onto the solid support followed by
225
solvent-exchange with aqueous buffer solution to promote bilayer formation. The selected lipid
226
composition was 70 mol% DOPC and 30 mol% POPG in order to prepare negatively charged
227
supported lipid bilayers which support MAC protein adsorption39. The QCM-D technique allows
228
label-free measurement of the mass and viscoelastic properties of the adsorbed material, enabling
229
detailed characterization of the fabricated lipid bilayer and each step of protein addition. After
230
forming the lipid bilayer, the final frequency and dissipation shifts are -25.0 ± 1.4 Hz and 0.6 ±
231
0.3 x 10-6, respectively, and the values are in agreement with past reported values39, 46, 47 (Figure
232
S2). An additional step of BSA protein addition to bare and bilayer-coated substrates indicated
233
that the bilayers are >95% complete and passivated remaining parts of the substrate in order to
234
specifically detect membrane association of MAC proteins to the lipid bilayer (Figure S3).
235
Thereafter, as part of the MAC assay described in Figure 1A, the change in frequency shift was
236
recorded as a function of time upon addition of protein components of the membrane attack
237
complex, with C5b-7, C8 and C9 proteins added in sequential format (Figure 1B).
238 239
Each stage of protein addition (indicated by the respective arrows) led to a negative frequency
240
shift. Because the frequency shift is negatively proportional to the adsorbed mass, the frequency
11 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 12 of 29
241
shifts indicate that the proteins attach to the lipid bilayer and the adsorbed mass of each protein
242
can be quantified (Figure 1C). By taking into account the molecular weights of each protein, the
243
molar ratio of bound proteins can be determined. Based on this approach, it
244 245
Figure 1. QCM-D Monitoring of MAC Assembly on the SLB Platform. (A) Schematic
246
illustration of MAC formation via sequential addition format. Changes in (B) frequency and (C)
247
Sauerbrey mass were monitored as functions of time after sequential addition of C5b-7 complex,
248
C8 protein, and C9 protein onto the 70:30 mol% DOPC:POPG SLB platform. The SLB on
249
silicon oxide was initially formed by the SALB method, and then the measurement time was
250
normalized. At t = 0 min, the MAC assay was initiated and the C5b-7 complex was added at t =
251
20 min. Then, C8 protein was added at t = 80 min and C9 protein at t = 150 min. The adsorbed
252
mass was calculated by the Sauerbrey relationship and the mass sensitivity constant for a 1Hz
253
frequency shift is 17.7 ng/cm2.
12 ACS Paragon Plus Environment
Page 13 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
254
was determined that approximately 1.25 C8 and 3.60 C9 proteins were attached to the lipid
255
bilayer per 1 C5b-7 protein. The calculated C5b-7:C8 molar ratio is in good agreement with
256
literature reports that indicate a 1:1 stoichiometry33. From these calculations, we also determined
257
that the measured stoichiometry of the C5b-8 intermediate complex to C9 protein is 3.4, which
258
agrees well with the literature range between 3 and 1627,
259
indicate that the platform offers a quantifiable, label-free assessment of bound protein mass,
260
leading us to investigate how the two inhibitors each affect MAC assembly.
33, 48, 49
. Taken together, the results
261 262
Effect of Vitronectin on MAC Assembly
263 264
Using the supported lipid bilayer platform, we first investigated the effect of vitronectin on
265
membrane association of the C5b-7 complex. The C5b-7 complex and vitronectin were mixed
266
before experiment, and then added to the lipid bilayer platform (Figure 2A). With increasing
267
vitronectin concentration, there was a general decrease in the frequency shift associated with
268
protein adsorption (Figure 2B). In order to investigate details of the vitronectin concentration-
269
dependent effects, the maximum frequency shifts are presented as a function of vitronectin
270
concentration in Figure 2C. Interestingly, at lower vitronectin concentrations (5 nM and below),
271
the frequency shifts were lower than the value obtained from the association of C5b-7 complex
272
in the absence of inhibitor. These results support that some of the C5b-7 complex interacted with
273
vitronectin in the solution and loses the ability to associate on the bilayer at lower vitronectin
274
concentration. That is, when vitronectin directly interacts with C5b-7 in solution, the resulting
275
vitronectin-C5b-7 complex is prevented from subsequently binding to the lipid bilayer. By
276
contrast, at higher vitronectin concentrations, there is a clear increase in the total adsorbed
13 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 14 of 29
277 278
Figure 2. Influence of Vitronectin on Membrane Association of the C5b-7 Complex. (A)
279
Schematic illustration shows addition of the C5b-7 complex which was preincubated with
280
vitronectin. Panel (B) presents the QCM-D frequency shift associated with C5b-7 + vitronectin
281
addition to the SLB platform as a function of vitronectin concentration. The C5b-7 complex
282
concentration was fixed at 20 nM. The proteins were added at t = 20 min. Panel (C) shows
283
absolute value of the maximum QCM-D frequency shift due to the amount of total adsorbed
284
protein as a function of vitronectin concentration based on the data presented in Panel (B). Each
285
data point is the average + standard deviation of three independent (n = 3) measurements. (D-E)
286
Corresponding schematic illustration and QCM-D measurement data for the addition of
287
vitronectin to the SLB platform, followed by addition of C5b-7 complex to the same platform.
288
Panel (F) shows absolute values of the maximum QCM-D frequency shifts due to the amounts of
289
adsorbed vitronectin and C5b-7 complex as a function of vitronectin concentration based on the
290
data presented in Panel (E). Each data point is the average + standard deviation of three
291
independent (n = 3) measurements. 14 ACS Paragon Plus Environment
Page 15 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
292
protein mass. In particular, at 100 nM and higher vitronectin concentrations, there are large
293
frequency shifts which suggest that vitronectin was the dominant adsorbed species because the
294
C5b-7 protein concentration was fixed at 20 nM.
295 296
In order to clarify the effect of vitronectin on C5b-7 adsorption, a sequential addition protocol
297
was followed in parallel experiments across the same range of inhibitor concentrations (Figure
298
2D). Figure 2E presents the individual frequency shifts associated with membrane association of
299
vitronectin followed by C5b-7 protein. Again, with increasing vitronectin concentration, there
300
was an increase in vitronectin uptake. On the other hand, increasing concentrations of vitronectin
301
inhibited subsequent membrane association of C5b-7. As presented in Figure 2F, the frequency
302
shifts were proportional to the vitronectin concentration ranging up to -19 Hz at 200 nM
303
concentration. Likewise, prior membrane association of vitronectin suppressed membrane
304
association of C5b-7, with negligible C5b-7 addition at high vitronectin concentrations.
305
Particularly strong inhibition was observed at vitronectin concentrations above 50nM. Taken
306
together, the data obtained from both experimental strategies support that vitronectin inhibits
307
membrane association of C5b-7 via direct interaction with C5b-7 which prevents binding to the
308
lipid bilayer and via competitive membrane association onto the supported lipid bilayer which
309
restricts the number of available adsorption sites.
310 311
Following addition of the i) premixed C5b-7 and vitronectin or ii) vitronectin followed by C5b-7,
312
there was next sequential addition of C8 and C9 proteins in order to investigate the effect of
313
surface-bound vitronectin. In Figure 3, the frequency shifts associated with C8 and C9 protein
15 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 16 of 29
314
addition are presented as a function of vitronectin concentration. Independent of the route by
315
which the supported lipid bilayer was treated with vitronectin, subsequent membrane association
316 317
Figure 3. Effect of Vitronectin on the Sequential Addition of C8 and C9 Proteins to the
318
SLB Platform. (A) Schematic illustration of sequential addition of C8 protein to the SLB
319
platform after vitronectin and C5b-7 addition. (B) QCM-D maximum frequency shift associated
320
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
Page 17 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
324
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
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 18 of 29
339
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).
18 ACS Paragon Plus Environment
Page 19 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
361
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
19 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 20 of 29
370
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
Page 21 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
393
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
21 ACS Paragon Plus Environment
51
that
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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.
Page 22 of 29
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
22 ACS Paragon Plus Environment
Page 23 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
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
23 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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
24 ACS Paragon Plus Environment
Page 24 of 29
Page 25 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
471
Biomacromolecules
REFERENECES
472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513
1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
12.
13. 14. 15.
16.
17. 18.
Ricklin, D.; Hajishengallis, G.; Yang, K.; Lambris, J. D., Complement: a key system for immune surveillance and homeostasis. Nat. Immunol. 2010, 11, 785-797. Rus, H.; Cudrici, C.; Niculescu, F., The role of the complement system in innate immunity. Immunol. Res. 2005, 33, 103-112. Ricklin, D.; Lambris, J. D., Therapeutic control of complement activation at the level of the central component C3. Immunobiology 2015, doi:10.1016/j.imbio.2015.06.012. Merle, N.; Church, S. E.; Fremeaux-Bacchi, V.; Roumenina, L. T., Complement system part I–molecular mechanisms of activation and regulation. Front. Immunol. 2015, 6. Morgan, B. P., The membrane attack complex as an inflammatory trigger. Immunobiology 2015, doi:10.1016/j.imbio.2015.04.006. Gros, P., In self-defense. Nat. Struct. Mol. Biol. 2011, 18, 401-402. Ratner, B. D.; Hoffman, A. S.; Schoen, F. J.; Lemons, J. E., Biomaterials science: an introduction to materials in medicine. Academic press: 2004. Molino, N. M.; Bilotkach, K.; Fraser, D. A.; Ren, D.; Wang, S.-W., Complement Activation and Cell Uptake Responses Toward Polymer-Functionalized Protein Nanocapsules. Biomacromolecules 2012, 13, 974-981. Nilsson, B.; Ekdahl, K. N.; Mollnes, T. E.; Lambris, J. D., The role of complement in biomaterial-induced inflammation. Mol. Immunol. 2007, 44, 82-94. Ratner, B. D., The catastrophe revisited: blood compatibility in the 21st century. Biomaterials 2007, 28, 5144-5147. Ekdahl, K. N.; Nilsson, B.; Gölander, C. G.; Elwing, H.; Lassen, B.; Nilsson, U. R., Complement activation on radio frequency plasma modified polystyrene surfaces. J. Colloid Interface Sci. 1993, 158, 121-128. Elwing, H.; Dahlgren, C.; Harrison, R.; Lundström, I., Complement factor adsorption on solid surfaces—an ellipsometric method for investigation of quantitative aspects. J. Immunol. Meth. 1984, 71, 185-191. Janatova, J.; Cheung, A.; Parker, C., Biomedical polymers differ in their capacity to activate complement. Complement Inflamm. 1990, 8, 61-69. Tengvall, P.; Askendal, A.; Ingemar, L., Complement activation by 3-mercapto-1, 2propanediol immobilized on gold surfaces. Biomaterials 1996, 17, 1001-1007. Zhang, Y.; Wang, C.; Hu, R.; Liu, Z.; Xue, W., Polyethylenimine-Induced Alterations of Red Blood Cells and Their Recognition by the Complement System and Macrophages. ACS Biomater. Sci. Eng. 2015, 1, 139-147. Appavu, R.; Chesson, C. B.; Koyfman, A. Y.; Snook, J. D.; Kohlhapp, F. J.; Zloza, A.; Rudra, J. S., Enhancing the Magnitude of Antibody Responses through Biomaterial Stereochemistry. ACS Biomater. Sci. Eng. 2015, 1, 601-609. Bhakdi, S.; Tranum-Jensen, J., Membrane damage by complement. Biochim. Biophys. Acta, Rev. Biomembr. 1983, 737, 343-372. McGeer, P.; Akiyama, H.; Itagaki, S.; McGeer, E., Activation of the classical complement pathway in brain tissue of Alzheimer patients. Neurosci. Lett. 1989, 107, 341-346.
25 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
19. 20.
21.
22. 23. 24. 25.
26.
27.
28.
29.
30.
31.
32.
33. 34.
Page 26 of 29
Bhakdi, S.; Tranum-Jensen, J., Molecular nature of the complement lesion. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5655-5659. Tschopp, J., Ultrastructure of the membrane attack complex of complement. Heterogeneity of the complex caused by different degree of C9 polymerization. J. Biol. Chem. 1984, 259, 7857-7863. Hadders, Michael A.; Bubeck, D.; Roversi, P.; Hakobyan, S.; Forneris, F.; Morgan, B. P.; Pangburn, Michael K.; Llorca, O.; Lea, Susan M.; Gros, P., Assembly and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and sC5b9. Cell Rep. 2012, 1, 200-207. Gilbert, R. J. C.; Serra, M. D.; Froelich, C. J.; Wallace, M. I.; Anderluh, G., Membrane pore formation at protein–lipid interfaces. Trends Biochem. Sci. 2014, 39, 510-516. Götze, O.; Müller-Eberhard, H. J., Lysis of erythrocytes by complement in the absence of antibody. J. Exp. Med. 1970, 132, (5), 898-915. Müller-Eberhard, H. J., The killer molecule of complement. J. Invest. Dermatol. 1985, 85, 47s-52s. Tschopp, J.; Chonn, A.; Hertig, S.; French, L., Clusterin, the human apolipoprotein and complement inhibitor, binds to complement C7, C8 beta, and the b domain of C9. J. Immunol. 1993, 151, 2159-2165. Preissner, K. T.; Podack, E.; Müller-Eberhard, H., The membrane attack complex of complement: relation of C7 to the metastable membrane binding site of the intermediate complex C5b-7. J. Immunol. 1985, 135, 445-451. Bhakdi, S.; Käflein, R.; Halstensen, T.; Hugo, F.; Preissner, K.; Mollnes, T., Complement S-protein (vitronectin) is associated with cytolytic membrane-bound C5b-9 complexes. Clin. Exp. Immunol. 1988, 74, 459. McDonald, J. F.; Nelsestuen, G. L., Potent inhibition of terminal complement assembly by clusterin: characterization of its impact on C9 polymerization. Biochemistry 1997, 36, 7464-7473. Sugita, Y.; Ito, K.; Shiozuka, K.; Suzuki, H.; Gushima, H.; Tomita, M.; Masuho, Y., Recombinant soluble CD59 inhibits reactive haemolysis with complement. Immunology 1994, 82, 34. Tschopp, J.; Masson, D.; Schaefer, S.; Peitsch, M.; Preissner, K. T., The heparin binding domain of S-protein/vitronectin binds to complement components C7, C8, and C9 and perforin from cytolytic T-cells and inhibits their lytic activities. Biochemistry 1988, 27, 4103-4109. Milis, L.; Morris, C.; Sheehan, M.; Charlesworth, J.; Pussell, B., Vitronectin-mediated inhibition of complement: evidence for different binding sites for C5b-7 and C9. Clin. Exp. Immunol. 1993, 92, 114. Bhakdi, S.; Tranum-Jensen, J., On the cause and nature of C9-related heterogeneity of terminal complement complexes generated on target erythrocytes through the action of whole serum. J. Immunol. 1984, 133, 1453-1463. Silversmith, R. E.; Nelsestuen, G. L., The fluid-phase assembly of the membrane attack complex of complement. Biochemistry 1986, 25, 841-851. Silversmith, R. E.; Nelsestuen, G. L., Interaction of complement proteins C5b-6 and C5b7 with phospholipid vesicles: effects of phospholipid structural features. Biochemistry 1986, 25, 7717-7725.
26 ACS Paragon Plus Environment
Page 27 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602
Biomacromolecules
35.
36. 37. 38. 39.
40.
41.
42.
43.
44.
45. 46.
47. 48. 49.
50.
Sheehan, M.; Morris, C.; Pussell, B.; Charlesworth, J., Complement inhibition by human vitronectin involves non-heparin binding domains. Clin. Exp. Immunol. 1995, 101, 136141. Bubeck, D., The making of a macromolecular machine: Assembly of the membrane attack complex. Biochemistry 2014, 53, 1908-1915. Mathern, D. R.; Heeger, P. S., Molecules great and small: the complement system. Clin. J. Am. Soc. Nephrol. 2015, CJN. 06230614. Ros, U.; García-Sáez, A. J., More than a pore: the interplay of pore-forming proteins and lipid membranes. J. Membr. Biol. 2015, 248, 545-561. Yorulmaz, S.; Tabaei, S. R.; Kim, M.; Seo, J.; Hunziker, W.; Szebeni, J.; Cho, N.-J., Membrane attack complex formation on a supported lipid bilayer: initial steps towards a CARPA predictor nanodevice. Eur. J. Nanomed. 2015, 7, 245-255. Steinmetz, N. F.; Bock, E.; Richter, R. P.; Spatz, J. P.; Lomonossoff, G. P.; Evans, D. J., Assembly of Multilayer Arrays of Viral Nanoparticles via Biospecific Recognition: A Quartz Crystal Microbalance with Dissipation Monitoring Study. Biomacromolecules 2008, 9, 456-462. Huang, C.-J.; Cho, N.-J.; Hsu, C.-J.; Tseng, P.-Y.; Frank, C. W.; Chang, Y.-C., Type I collagen-functionalized supported lipid bilayer as a cell culture platform. Biomacromolecules 2010, 11, 1231-1240. Tseng, P.-Y.; Chang, Y.-C., Tethered Fibronectin Liposomes on Supported Lipid Bilayers as a Prepackaged Controlled-Release Platform for Cell-Based Assays. Biomacromolecules 2012, 13, 2254-2262. Reviakine, I.; Rossetti, F. F.; Morozov, A. N.; Textor, M., Investigating the properties of supported vesicular layers on titanium dioxide by quartz crystal microbalance with dissipation measurements. J. Chem. Phys 2005, 122, 204711. Cho, N.-J.; Kanazawa, K. K.; Glenn, J. S.; Frank, C. W., Employing two different quartz crystal microbalance models to study changes in viscoelastic behavior upon transformation of lipid vesicles to a bilayer on a gold surface. Anal. Chem. 2007, 79, 7027-7035. Sauerbrey, G., Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung. Z. Physik 1959, 155, 206-222. Tabaei, S. R.; Jackman, J. A.; Kim, S.-O.; Zhdanov, V. P.; Cho, N.-J., Solvent-Assisted Lipid Self-Assembly at Hydrophilic Surfaces: Factors Influencing the Formation of Supported Membranes. Langmuir 2015, 31, 3125-3134. Tabaei, S. R.; Choi, J.-H.; Haw Zan, G.; Zhdanov, V. P.; Cho, N.-J., Solvent-Assisted Lipid Bilayer Formation on Silicon Dioxide and Gold. Langmuir 2014, 30, 10363-10373. Kolb, W. P.; Haxby, J. A.; Arroyave, C. M.; Müller-Eberhard, H. J., Molecular analysis of the membrane attack mechanism of complement. J. Exp. Med. 1972, 135, 549-566. Stewart, J. L.; Monahan, J. B.; Brickner, A.; Sodetz, J. M., Measurement of the ratio of the eighth and ninth components of human complement on complement-lysed membranes. Biochemistry 1984, 23, 4016-4022. Chauhan, A.; Moore, T., Presence of plasma complement regulatory proteins clusterin (Apo J) and vitronectin (S40) on circulating immune complexes (CIC). Clin. & Exp. Immunol. 2006, 145, 398-406.
27 ACS Paragon Plus Environment
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
603 604 605 606 607 608 609 610 611
51.
52.
Podack, E. R.; Müller-Eberhard, H. J., Binding of desoxycholate, phosphatidylcholine vesicles, lipoprotein and of the S-protein to complexes of terminal complement components. J. Immunol. 1978, 121, 1025-1030. Briand, E.; Zäch, M.; Svedhem, S.; Kasemo, B.; Petronis, S., Combined QCM-D and EIS study of supported lipid bilayer formation and interaction with pore-forming peptides. Analyst 2010, 135, 343-350.
612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 28 ACS Paragon Plus Environment
Page 28 of 29
Page 29 of 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
637
Biomacromolecules
TABLE OF CONTENTS (TOC) GRAPHIC
638
639
29 ACS Paragon Plus Environment