SP-B

1
Nanostructure Changes in Lung Surfactant
Monolayers Induced by Interactions between
Palmitoyloleoylphosphatidylglycerol and
Surfactant Protein BJunqi Ding, Ivo Doudevski,
Heidi E. Warriner, Timothy Alig, and Joseph A.
Zasadzinski
2
What does a lung surfactant do?

• Forms a monolayer
• Lowers surface tension upon compression
• Respreads quickly on expansion
• Reduces the work of breathing
• Prevents illnesses of the lungs

3
Surface Tension

• Tendency of molecules to be pulled toward the
  center
• Surface tension of pure water is about 70mN/m
• Surface tension of lung surfactant is near 0
• The higher the surface tension, the more the
  monolayer would like to maintain small area
• Low surface tension in monolayer is critical

http//www.quest.arc.nasa.gov
4
What are the components in LS?

• A complex mixture of lipids and proteins
• Consists primarily of saturated
  dipalmitoylphosphatidylcholine (DPPC)
• Smaller fractions of unsaturated
  phosphatidylcholines (PCs)
• Anionic phospholipids, such as phospatidylglycerol
  s (PGs)
• Anionic lipids, such as palmitic acid (PA)
• Neutral components, such as cholesterol
• 4 lung surfactant-specific proteins

5
Lung surfactant proteins

• SP-A and SP-D
• Larger proteins
• Responsible for host defense mechanisms
• Aid in transport and recycling of lung surfactant

• SP-B and SP-C
• Smaller proteins
• Intensely hydrophobic
• Important to surface activity

6
Why is this complex mixture so complex?

• Conflicting requirements of surfactant
• Multiple lipids and proteins are necessary
• DPPC alone forms rigid monolayers but can not
  adsorb or respread quickly
• Unsaturated PGs are fluid enough and fast
  spreading, but can not lower surface tension
  sufficiently
• Monolayer collapse is also a factor in this
  complexity

7
Monolayer Collapse

• The maximum pressure sustained is set by the
  instability known as monolayer collapse
• Monolayer collapse is the point at which the
  surfactant breaks
• The surfactant must respread quickly in order to
  continue breathing
• Proteins and lipids work together in order to
  continue with normal breathing

8
Squeeze-out Theory

• Most of the unsaturated and anionic lipids in the
  LS monolayer have a low collapse pressure
  relative to DPPC
• The selective removal of lipids with low collapse
  pressures occurs as pressure increases
• Low collapse pressure components are squeezed out
  of the monolayer
• DPPC continues to lower the surface tension near
  zero
• Squeezed out lipids are reincorporated into the
  monolayer upon expansion
• Surfactant proteins aid in this reincorporation

Ka Yee C. Lee, et al. Biophysical Journal 2001,
81, 572-585
9
Compression-Expansion-Compression Isotherms of
DPPG Monolayer
Takamoto, et al. Biophysical Journal 2001, 81,
153-169
10
Monolayer Collapse
www.nsr.bioeng.washington.edu
11
Why is it important?

• Respiratory distress syndrome (RDS) occurs in
  premature babies carried less than 34 weeks
• The immature lung lacks the necessary surfactant
  in order to keep their lungs from collapsing
• It is a serious lung condition that affects
  40,000 infants in the US each year, resulting in
  thousands of deaths

http//www.lungusa.org
12
Not Convinced Yet????

• Cystic fibrosis (CF)
• CF patients have a decrease of SP-A
• Since SP-A is responsible for bacterial defense,
  its loss may increase the possibility of lung
  infection in patients
• Alterations in surfactant lipid composition in
  patients impairs the surface tension lowering
  function
• Pneumonia
• Patients with pneumonia have a reduced PC and PG
  content, leading to the impairment of the surface
  tension lowering function
• The amount of SP-A is decreased, causing patient
  host defense properties to lower

http//respiratory-research.com/
13
Smoke

• Smoking alters surfactant compostion and function
• Levels of SP-A and SP-D are decreased, which can
  contribute to the increased incidence of
  respiratory infections
• Cigarette smokes nitrites and oxidants can
  inactivate alpha-1-proteinase inhibitor, which is
  responsible for preventing the breakdown of
  tissue in the lungs, causing negative effects on
  surfactant function

http//respiratory-research.com/
14
2 Surfactant Replacement Therapies

• Exosurf is a synthetic formulation that contains
  80 DPPC with hexadecanol and tyloxapol
• DPPC is found in natural LS but the other 2
  components are not
• Survanta is an extract of bovine lung surfactant
  that contains SP-B and SP-C and is supplemented
  with PA, triglycerides, and DPPC
• The properties exhibited by Survanta can provide
  a benchmark for new synthetic replacement
  surfactants

http//www.sciencedaily.com http//www.survanta.co
m
15
Need for New Surfactant Replacement Therapy

• Surfactant replacement therapy has reduced
  mortality rates by 30-50 for infants with RDS
• Animal sources are difficult
• expensive to purify
• risk of containing contaminants
• lack consistency between batches
• Currently, surfactant replacement therapies can
  not used to treat diseases of the mature lung in
  adults

http//www.survanta.com Frank Bringezu, et al.
Langmuir 2001, 17, 4641-4648
16
Research Limitations

• Lack of fundamental understanding of
• The roles of the individual components of LS
• The way components interact in the monolayer and
• Their affect on monolayer collapse and
  respreading
• A better understanding is necessary in order to
  design new synthetic replacement therapies that
  can be tailored for treatment of different
  diseases and illnesses

17
Nanostructure Changes in Lung Surfactant
Monolayers Induced by Interactions between
Palmitoyloleoylphosphatidylglycerol and
Surfactant Protein BJunqi Ding, Ivo Doudevski,
Heidi E. Warriner, Timothy Alig, and Joseph A.
Zasadzinski
18
Abstract

• Langmuir isotherms, Brewster angle microscopy
  (BAM), and Atomic force microscopy AFM will be
  used to try and replicate some of the properties
  Survanta exhibits
• They will use a synthetic dimeric peptide based
  on the SP-B protein to determine its possible use
  in LS replacement therapies
• DPPC/POPG/PA monolayers will be studied both
  before and after the addition of the dSP-B and
  compared to the results found with Survanta
  monolayers

19
Langmuir Trough for measuring Pressure-Area
Isotherms
Ising.phys.cwru.edu/surfactants/measurement.html
20
Pressure-Area Isotherm
www.abo.fi/fak/mnf/fysik/mole/LB.html
21
AFM

• Contact mode AFM is used to view samples that are
  too small to see under the optical microscope
• Provides details of the morphology
• Tip scans the surface in order to get height
  profiles

22
Brewster Angle Microscopy

• Uses an argon laser as a light source
• Mirror and polarizer are placed between the laser
  and the trough
• Provides light at the Brewster angle (53.1o)
• Additional polarizer and analyzer improve
  contrast and determine changes in molecular tilt
  angle
• Contrast is due to local differences in monolayer
  refractive index caused by molecular density or
  packing
• Useful to follow morphology in absence of any
  fluorescence dye

Henson, S Meunier, J. Rev. Sci. Instrum. 1991,
62, 936-939
23
The Importance of SP-B in LS

• SP-B is the only protein necessary for postnatal
  lung function and survival
• Possibly responsible for the prevention of
  squeeze-out
• 78-residue, lipid associating protein
• Can the synthetic peptide prove useful as a
  replacement?

24
SP-B
B dSP-B1-25 mimetic peptide
C molecular surface representation
A hypothetical structure of native SP-B
homodimer
N-terminal domain is shown in purple,
midsequences are shown in blue, C-termial
sequence in green, and the disulfide connectivity
in yellow.
Ding, et al. Langmuir 2003, 19, 1539-1550
25
Survanta Isotherm at 25oC
A plateau occurs around a surface pressure of
40mN/m.
Ding, et al. Langmuir 2003, 19, 1539-1550
26
AFM Images of Survanta Monolayers Before and
After Plateau
C,E Surface pressure of 45mN/m
B,D Surface pressure of 30mN/m
Nanosilos are seen in the fluid phase
Ding, et al. Langmuir 2003, 19, 1539-1550
27
What are These Nanosilos?

• Nanosilos are lipid-protein structures from
  40-300 nm in diameter and 5-8 nm in height
• Nanosilos may be a mechanism of stabilizing the
  unsaturated lipids and SP-B protein in the
  vicinity of the monolayer for subsequent
  reincorporation as the monolayer is expanded
• Are they only an intrinsic feature of Survanta?
• Do they only occur above the plateau?
• Is it possible to replicate these nanosilos and
  determine what causes their formation?

28
Isotherms of DPPC/POPG/PA
Ding, et al. Langmuir 2003, 19, 1539-1550
29
Isotherms of DPPGPOPG Monolayers
Takamoto, et al. Biophysical Journal 2001, 81,
153-169
30
Isotherms of DPPC/POPG/PA with dSP-B1-25
Ding, et al. Langmuir 2003, 19, 1539-1550
31
BAM Images of MA
Increasing POPG content
Increasing DPPC content
Ding, et al. Langmuir 2003, 19, 1539-1550
32
BAM Images of MB
Increasing POPG content
Increasing DPPC content
Ding, et al. Langmuir 2003, 19, 1539-1550
33
AFM Images Of MA2 and MB2
MA2 at 30mN/m at 25oC
MA2 at 40mN/m at 25oC
MB2 at 30mN/m at 25oC
MA2 (DPPC/POPG/PA50/40/8)
MB2 (DPPC/POPG/PA/dSP-B1-2550/40/8/10)
Ding, et al. Langmuir 2003, 19, 1539-1550
34
AFM of DPPC/POPG/PA/dSP-B1-25 Mixtures Deposited
at 40mN/m at 25oC
60/30/8/10
70/20/8/10
50/40/8/10
80/10/8/10
Ding, et al. Langmuir 2003, 19, 1539-1550
35
AFM of Various Lipid Mixtures with dSP-B1-25
DPPC/PA/dSP-B1-25 75/8/10
DPPC/POPG/dSP-B1-25 75/15/10
DPPC/dSP-B1-25
POPG/dSP-B1-25
PA/dSP-B1-25
Ding, et al. Langmuir 2003, 19, 1539-1550
36
AFM of DPPC/POPG/PA (75/15/8) with varying
dSP-B1-25
0 d-SP-B1-25
2.5 d-SP-B1-25
5 d-SP-B1-25
7.5 d-SP-B1-25
10 d-SP-B1-25
Ding, et al. Langmuir 2003, 19, 1539-1550
37
AFM Images of DPPC/POPG/PA/dSP-B1-25 (75/15/8/10)
on Freshly Cleaved Mica and Oxidized Silica Wafers
Ding, et al. Langmuir 2003, 19, 1539-1550
38
Discussion

• Individual components of LS are either good at
  lowering surface tension or fluidizing the
  monolayer, no single lipid or protein exhibits
  both properties
• Current thought is that lipids and proteins that
  are squeezed out from the monolayer occupy a
  surface-associated reservoir near the interface
• From AFM images , it appears that nanosilos are
  part of this surface-associated reservoir

39
Conclusions

• Plateaus in the isotherms occur only for SP-B
  containing monolayers and the extent of the
  plateaus depend on the concentration of POPG
• Nanosilos are present above the plateau pressure
  in monolayers containing SP-B and POPG
• Nanosilos are present in both Survanta and the
  model synthetic surfactant with the SP-B peptide

40
Conclusions Contd

• If POPG is squeezed out from the monolayer
  without being held in a nanosilo, it is likely
  that it does not reincorporate on expansion
• Nanosilos retain both protein and fluid lipid in
  the immediate vicinity of the monolayer at high
  surface pressure and allows both molecules to
  reincorporate in the monolayer at a lower
  pressure
• Model lung surfactant mixture and synthetic
  peptide capture the morphologies present in the
  bovine extract Survanta that contains the native
  SP-B