Structure of Lung-Mimetic Multilamellar Bodies with Lipid

May 30, 2018 - The Structure of Lung-mimetic Multilamellar Bodies with Lipid Compositions Relevant in Pneumonia. Dylan Steer , Sherry Leung , Hannah ...
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Biological and Environmental Phenomena at the Interface

The Structure of Lung-mimetic Multilamellar Bodies with Lipid Compositions Relevant in Pneumonia Dylan Steer, Sherry Leung, Hannah Meiselman, Daniel Topgaard, and Cecilia Leal Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01359 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 30, 2018

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a)

3:1 DPPC:DOPG

Intensity [arb. u.]

Low CL High CL

b)

4:1 DPPC:DOPG Low CL High CL

"diseased"

"diseased"

"healthy"

"healthy"

0.0 0.1 0.2 0.3 0.4 0.0 0.1 0.2 0.3 0.4 q [Å-1] q [Å-1] 12 9 6 3 0

mol% CL

c)

6

8 10 12 14 q [10-2 Å-1]

6

8 10 12 14 q [10-2 Å-1]

Intensity 100

d)

102

101

170

d-spacing [Å]

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

Langmuir

3:1 4:1

150

DPPC:DOPG one phase two phase

130 110

e)

90

0 3 6 9 12 Cardiolipin (CL) [mol %]

Type I

Type II or

f) height 1

height 2

5µm

Low mobility

High mobility

Mixed Lipids

FIGURE 1 FIGURE 1 ACS Paragon Plus Environment

Langmuir

b)

High Ca2+

Low CL 0.08

0.10 0.12 q [Å-1]

High Ca2+

0.14

c) 140

Low Ca2+

130

Low CL

d-spacing [Å]

Intensity [arb. u.]

a) Low Ca2+ Intensity [arb. u.]

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

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120 110 100

High CL 0.08

0.10 0.12 q [Å-1]

0.14

FIGURE 2

ACS Paragon Plus Environment

90

High CL 0

3

6 9 [Ca ] [mM] 2+

12

a)

Low Ca2+. 4:1 DPPC: DOPG raw data liquid-like gel-like full fit

Intensity [Arb. Units]

1.0

b)

Intensity [Arb. Units]

1.2

1.4

1.8

2.0

High Ca2+ Low Ca2+

1.2

1.4

1.6

q [Å-1] High CL. 4:1 DPPC: DOPG

1.8

2.0

High Ca2+ Low Ca2+

1.0

1.2

1.4

d) 3:1 DPPC:DOPG

1.3

1.6

q [Å-1]

Low CL. 4:1 DPPC: DOPG

1.0

c)

High CL Low CL

Intensity [arb. u.]

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

Langmuir

Intensity [Arb. Units]

Page 3 of 47

1.6

q [Å-1]

CL

Low Ca2+

Low Ca2+ 1.5 1.6 q [Å-1]

2.0

e) 4:1 DPPC:DOPG

CL

1.4

1.8

1.3

1.4

1.5 1.6 1.7 q [Å-1]

FIGURE 3

ACS Paragon Plus Environment

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a) unsaturated g β g2 carbons

130

g3

gα β

70

g1 gγ

65 13

b) ω1

ω2

c3

ω3

g2 O g g3

O

O

1

O

ω1 ω3

c10 c9

ω2

c3

O

c2

O

O

g1 O

g3

g2 O

β

O P O O-

O P O-

γ

60 55 C chemical shift [ppm]

DPPC

O

c4 c2

α

DP CP INEPT

α

+ N

γ

gα gγ O gβ OH

γ γ

CH2 groups c2

35

ω2 ω1 ω 3

DOPG

OH

FIGURE 4

ACS Paragon Plus Environment

c13c12 c10 c9

c8/11 (DO) c3

ω3

30

c3

ω2

25

Cardiolipin (CL) O

c2

O O

g1 g2 O

O

g3 gO αc gβcOH O

O O

O O P O

O P O O

O

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a) Low Ca2+. 3:1 DPPC: DOPG ω3 ( )

CH2 ( )

CL 15

CL 15

CL 0 72

71 70 65 δ13C [ppm]

64 63 δ13C [ppm]

62

c) Low CL. 3:1 DPPC: DOPG g2 g 3( ) g 1 ( )

CL 0 33

32 31 δ13C [ppm]

ω3 ( )

CH2 ( )

62

e) High CL. 3:1 DPPC: DOPG g2 g 3( ) g 1 ( )

Ca 12 Ca 6 Ca 0 33

32 31 δ13C [ppm]

ω3 ( )

δ13C [ppm]

64 δ13C [ppm]

63

62

f)

Ca 6

Ca 0 70 65

0.0 0.5 0

3

Ca 0 33

32

31

6 9 CL [mol%]

12

30

δ13C [ppm]

FIGURE 5

ACS Paragon Plus Environment

Low CL

0.5 0.0 0.5 fast

Ca 12

Ca 6

71

30

CH2 ( )

Ca 12

72

0.5

slow

Ca 0 64 63 δ13C [ppm]

Low Ca2+

d)

Ca 6

71 70 65 δ13C [ppm]

slow

fast

30

Ca 12

72

|CP/INEPT| signal

g 3( ) g 1 ( )

|CP/INEPT| signal

g2

b)

0

3

slow

|CP/INEPT| signal

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

Langmuir

6 9 Ca2+ [mM]

12

High CL

0.5 0.0 0.5 fast

0

3

6 9 Ca2+ [mM]

12

Langmuir 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

a) i)

ii) air

hypophase

air-water interface iii)

aveoli cells

Lung lipid multilamellar vesicles

b) CL

H2O

d

d

c) ii

iii

i

iv

R e p e at

FIGURE 6

ACS Paragon Plus Environment

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Langmuir

1

The Structure of Lung-mimetic Multilamellar Bodies with Lipid

2

Compositions Relevant in Pneumonia

3

Dylan Steera, Sherry Leunga, Hannah Meiselmana, Daniel Topgaardc, Cecilia

4

Leala,b,*

5

a

Department of Materials Science and Engineering, bFrederick Seitz Materials Research Laboratory. University of Illinois at Urbana-Champaign, Urbana, IL 61801

6 7

c

Division of Physical Chemistry, Center of Chemistry and Chemical Engineering, Lund

8

University. SE-221 00 Lund, Sweden

9

*Email: [email protected]

10

Keywords: multilamellar bodies, lung lipid, solid-state NMR, SAXS, cardiolipin

11

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12

Abstract

13

The hierarchical assembly of lipids, as modulated by composition and environment, plays a

14

significant role in the function of biological membranes and a myriad of diseases. Elevated

15

concentrations of calcium ions and cardiolipin, an anionic tetra-alkyl lipid found in mitochondria

16

and some bacterial cell membranes, have been implicated in pneumonia recently. However, their

17

impact on the physicochemical properties of lipid assemblies in lungs and how it impairs alveoli

18

function is still unknown. We use Small- and Wide- Angle X-ray Scattering (S/WAXS) and

19

Solid-State Nuclear Magnetic Resonance (ssNMR) to probe the structure and dynamics of lung-

20

mimetic multilamellar bodies (MLBs) in the presence of Ca2+ and cardiolipin. We conjecture that

21

cardiolipin overexpressed in the hypophase of alveoli strongly affects the structure of lung-lipid

22

bilayers and their stacking in the MLBs. Specifically, S/WAXS data revealed that cardiolipin

23

induces significant shrinkage of the water-layer separating the concentric bilayers in

24

multilamellar aggregates. ssNMR measurements indicate that this inter-bilayer tightening is due

25

to undulation repulsion damping as cardiolipin renders the glycerol backbone of the membranes

26

significantly more static. In addition to MLB dehydration, cardiolipin promotes intra-bilayer

27

phase separation into saturated-rich and unsaturated-rich lipid domains that couple across

28

multiple layers. Expectedly, addition of Ca2+ screens the electrostatic repulsion between

29

negatively charged lung membranes. However, when cardiolipin is present, addition of Ca2+

30

results in an apparent inter-bilayer expansion likely due to local structural defects. Combining

31

S/WAXS and ssNMR on systems with compositions pertinent to healthy and unhealthy lung

32

membranes, we propose how alteration of the physiochemical properties of multilamellar bodies

33

can critically impact the breathing cycle.

34

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Langmuir

35

Introduction

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Lungs critically rely on a hydrated lung lipid (often referred to as “surfactant”) layer (LS) present

37

at the interface between the pulmonary cells and the air to allow sufficient gas exchange, to

38

protect cells from contaminants and dehydration, and to modulate the surface energy of this

39

interface. The LS is composed of approximately 90% lipids and 10% proteins and coats the

40

interior of the alveolar sacks used in gas exchange with the blood stream.1, 2 In the process of

41

breathing-out the alveoli collapse and consequently shrink the exposed area of the mucus LS

42

coating. To allow re-inflation of the alveoli the LS surface must be expanded again, which

43

requires energy input postulated to be proportional to the surface tension of the coating.2 It is

44

generally accepted that perturbations of the LS coating lead to a number of health conditions due

45

to an unbalanced surface tension in alveoli.2, 3, 4, 5

46

The current understanding of LS structure and function has been acquired for decades and the

47

majority of studies model LS as a molecular interface between water and air.1, 4, 6, 7, 8 However,

48

LS is a sub-micron thick layer known to self-assemble into complex and heterogeneous 3D

49

structures.2,

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hydrophobic tails facing towards the air and the hydrophilic headgroups immersed in the

51

hydrated hypophase. In the hypophase there is a mixture of structures including multilamellar

52

bodies –concentric shells of lipid bilayers aligned like onion layers – and tubular myelin –

53

crossed three dimensionally networked patterns of lipid bilayers.2 The role of this complex phase

54

behavior in LS functionality is often overlooked but it is expected to critically impact LS

55

performance in healthy as well as in diseased states.11

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While the specific mechanism of action for LS continues to be an active area of research, studies

57

focused on thin lipid monolayers reveal that during lung compression the increased lateral

4, 9, 10

At the surface of the LS film there is a monolayer of lipids aligned with

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pressure selectively concentrates dipalmitoyl phosphatidylcholine (DPPC) lipids at the air-water

59

interface as a way to minimize surface energy.1,

60

having fully saturated chains with sixteen carbons and at physiologic temperatures packs very

61

densely with medium length scale ordering of parallel alkyl tails.13 With inhalation, the

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multilamellar bodies (MLBs) resupply the interface with unsaturated lipids as the surface area

63

increases in order to increase the fluidity and allow respreading of the film over the alveolar

64

walls2, 14. Several studies investigate the importance of lipid composition, dynamics, packing,

65

and structure on the LS-air interface, however the structure of the hypophase is relatively

66

unexplored, despite its importance.1,

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composition need to be considered for tubular myelin and multilamellar bodies extending beyond

68

minimal air/liquid interface models.

69

Pneumonia is the inflammation of pulmonary tissue due to infection that can lead to severe

70

breathing complications in terrestrial and marine mammals. Particularly, pneumonia is the

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leading cause of morbidity in dolphin populations.17 While pneumonia is a well-studied

72

pathology, there is currently an incomplete understanding of the role of lung lipids in this

73

diseased state. It has been observed recently that addition of heart-derived cardiolipin and an

74

increase in Ca2+ in healthy animal model lungs are intimately related to appearance of

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pneumonia-like symptoms.18,

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pulmonary illness were observed to present abnormally high cardiolipin levels in lung lipid

77

extracts (10-15 mol% of total lipids) compared to healthy mice (