Transcript SP-B - Louisiana State University

Nanostructure Changes in Lung
Surfactant Monolayers Induced by
Interactions between
Palmitoyloleoylphosphatidylglycerol
and Surfactant Protein B
Junqi Ding, Ivo Doudevski, Heidi E. Warriner,
Timothy Alig, and Joseph A. Zasadzinski
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
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
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 phospatidylglycerols
(PGs)
– Anionic lipids, such as palmitic acid (PA)
– Neutral components, such as cholesterol
– 4 lung surfactant-specific proteins
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
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
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
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
Compression-ExpansionCompression Isotherms of DPPG
Monolayer
Takamoto, et al. Biophysical Journal 2001, 81, 153-169
Monolayer Collapse
www.nsr.bioeng.washington.edu
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
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/
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 smoke’s 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/
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.com
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
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
Nanostructure Changes in Lung
Surfactant Monolayers Induced by
Interactions between
Palmitoyloleoylphosphatidylglycerol
and Surfactant Protein B
Junqi Ding, Ivo Doudevski, Heidi E. Warriner,
Timothy Alig, and Joseph A. Zasadzinski
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
Langmuir Trough for measuring
Pressure-Area Isotherms
Ising.phys.cwru.edu/surfactants/measurement.html
Pressure-Area Isotherm
www.abo.fi/fak/mnf/fysik/mole/LB.html
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
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
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?
SP-B
A: hypothetical structure
of native SP-B homodimer
B: dSP-B1-25 mimetic
peptide
C: molecular surface
representation
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
Survanta Isotherm at
o
25 C
A plateau occurs around a
surface pressure of 40mN/m.
Ding, et al. Langmuir 2003, 19, 1539-1550
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
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?
Isotherms of DPPC/POPG/PA
Ding, et al. Langmuir 2003, 19, 1539-1550
Isotherms of DPPG:POPG
Monolayers
Takamoto, et al. Biophysical Journal 2001, 81, 153-169
Isotherms of DPPC/POPG/PA
with dSP-B1-25
Ding, et al. Langmuir 2003, 19, 1539-1550
BAM Images of MA
Increasing
POPG content
Increasing
DPPC content
Ding, et al. Langmuir 2003, 19, 1539-1550
BAM Images of MB
Increasing
POPG content
Increasing
DPPC content
Ding, et al. Langmuir 2003, 19, 1539-1550
AFM Images Of MA2 and MB2
MA2 at 30mN/m at 25oC
MA2:
(DPPC/POPG/PA=50/40/8)
MA2 at 40mN/m at 25oC
MB2 at 30mN/m at 25oC
MB2:
(DPPC/POPG/PA/dSP-B1-25=50/40/8/10)
Ding, et al. Langmuir 2003, 19, 1539-1550
AFM of DPPC/POPG/PA/dSP-B1-25
Mixtures Deposited at 40mN/m at
25oC
50/40/8/10
60/30/8/10
70/20/8/10
Ding, et al. Langmuir 2003, 19, 1539-1550
80/10/8/10
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
PA/dSP-B1-25
Ding, et al. Langmuir 2003, 19, 1539-1550
DPPC/dSP-B1-25
POPG/dSP-B1-25
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
AFM Images of DPPC/POPG/PA/dSPB1-25 (75/15/8/10) on Freshly Cleaved
Mica and Oxidized Silica Wafers
Ding, et al. Langmuir 2003, 19, 1539-1550
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
Conclusions
• Plateaus in the isotherms occur only for SPB 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 SPB peptide
Conclusions Cont’d
• 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