Role of Oligo- and Polysaccharides in Modulationof Biological Interfaces

Motomu Tanaka
Biophysical Chemistry Laboratory II
Institute of Physical Chemistry andBIOQUANT, University of Heidelberg

ContentsMotivations, Scientific Background
Models of Cell-ECM Contacts: Polymer-Supported Membranes
Interactions Mediated via “Membrane-Bound” Sugars (Glycocalyx)
Molecular Mechanism of Bacterial Resistance to CAPs

Fundamental Motivation
How does Nature Regulate Cell-Cell (Cell-Tissue)
Interactions?

Alberts “Molecular Biology of the Cells”

Many contacts between two neighboring cells are mediated via
layers of hydrated biopolymers containing saccharide moieties(ECM and glycocalyx).

Design of defined cell membrane models with less complexity canreveal how biopolymers modulate interfacial interactions.

Cell-Cell Contact as “Wetting Problems” (I)Stratified layers are stable only if “complete wetting”
conditions are fulfilled at the interface.

The presence of an “additional” layer (membrane) must result in thegain of the surface free energy.
Spreading coefficient: S = σSL – (σSP + σPM + σML) > 0Brochard, de Gennes, Adv. Colloid Interf. Sci. 1992

Cell-Cell Contact as “Wetting Problems” (II)To keep a finite intercellular distance of 10 ~ 100 nm, theinteraction potential at the interface should be weakly repulsive.
electrostatic
van der Waals
hydration
entropic

Deq

Disjoining pressure (net force per unit area)

P ( D) = -¶G / ¶D

Churaev, Derjaguin, J. Colloid Interf. Sci. 1985
Tanaka et al., J. Phys. Cond. Matter 2005

22¶
G¶
D>0The minimum P ( Deq ) = 0 can be found at

Negative disjoning pressure causes dissipation (de-wetting).

Conventional Strategy
Use of lipid membranes on planar substrates (solidsupported membranes) as model biomembranes
A. Brian, H. McConnell PNAS 1985, E. Sackmann, Science 1996
bulk watermembranewater reservoir(d = 5 ~ 20 Å)

solid substrate

Human platelet integrin αIIbβ3 receptors
in a solid-supported membrane
Problem: Proteins in the membrane experience
direct mechanical contact to hard/solid substrates,

resulting in partial/complete denaturing.
Gönnenwein, Tanaka, Hu, Moroder, Sackmann, Biophys. J. 2003
Purrucker, Gönnenwein, Förtig, Rusp, Moroder, Sackmann, Jordan, Tanaka, SoftMatter 2007

Our Strategy: “Polymer-Supported Membrane” Concept
Use of hydrated polymer films as biocompatible interlayers thatmimic generic roles of cytoskeleton and extracellular matrix
G. Wegner (MPI Mainz)

Tanaka & Sackmann, Nature, 437, 656 (2005)

regenerated cellulose
thickness: 5 – 50 nm

The presence of ultrathin (d ~ 10 nm) polymer films fascilitates
an improved homogeneity and lateral diffusivity oftransmembrane cell receptors, retaining their natural functions.
Gönnenwein, Tanaka, Hu, Moroder, Sackmann, Biophys. J. (2003)
Purrucker, Gönnenwein, Förtig, Rusp, Moroder, Sackmann, Jordan, Tanaka
ChemPhysChem (2004), SoftMatter (2007), Phys. Rev. Lett. (2007)

A Breakthrough: Spreading of Native Cell Membranes
If it works, it would enable to retain the natural composition/density of proteins.

First Attempt: Human Red Blood Cell Membranes (Erythrocytes)
S. Kaufmann“Inside labeling” on a polymer support?
Orientation of cells is identified
with antibodies after spreading• No adhesion or rupture happens on glass/quartz.
On polymer supports:•“Inside labeling” results in a homogeneous and continuous signal.
• “Outside labeling” shows no fluorescence.

Complete Wetting of Cell Membranes
After spreading, membranes take “inside-out” orientation.

Transformation of 3D cells into quasi 2D films with the perfectly defined
membrane orientation→ First Example of “Two-Dimensional Cell Membranes”
The same principle works for other native membranes (microsomes, sarcoplasmic
reticulum, and plasma membrane extracts).
Tanaka, Kaufmann, Nissen, Hochrein, Phys. Chem. Chem. Phys. 2002
Tanaka, Wong, Rehfeldt, Tutus, Kaufmann, J. Am. Chem. Soc. 2004
Tanaka, Rossetti, Tutus, Schneck, Kaufmann, Weiss J. Struct. Biol. 2009.

Structure of Two-Dimensional Cell Membranes
Specular Neutron and X-ray Reflectivity
First X-ray Reflectivity Result of Human Red Blood Cell Membranes
on Polymer Support

F. Rossetti (DFG fellow)
E. Schneck, S. Kaufmann

Measured at ESRF ID10B at 22 keV,
cytoskeleton (d ~ 3.1 nm)

where the transmittance of X-ray
through 1 mm thick water is > 40%

membrane core (d ~ 3.6 nm)

O. Konovalov (ESRF)
The best fit model indicates theglycocalyx (d ~ 2.4 nm) +hydrated cellulose (d ~ 8.1 nm)

deposition of a uniform “layer” with aclear electron density contrast.

In parallel, we have been carrying out specular neutron reflectivity experiments at
ILL to highlight/mask a certain layer by contrast variation.
G. Fragnetto (ILL)Roles of Biopolymers in Fine-Adjustment ofInterfacial Interactions
Π(D) = 0 at equilibrium cell-cell (cell-substrate) distance Deq
vdW (asymmetric 5-slab model)
A121 A343
A545 A323
A545 A121
1 A234+ΠvdW =][ 3 3336p D( D + T2 )( D + T4 )( D + T2 + T4 )Helfrich Repulsion
Hydration Repulsion

P Helf3 p 2 ( kT ) 2=64 k D 3P hydD 
= P0 exp   l0 

All parameters to calculate three major forces can bemeasured/calculated quantitatively.

Comparison of Theoretically Predicted Deq and Dexp
500

Πhyd (thin)Πhyd (thick)

300
100

VdW
-100 0

100

200

300

400

500

600

700

D (A)

800

900

1000

Helfrich
P(10 LB)
P(30 LB)-300

VdW + Helfrich-500

Vdw + Helfr.+ Hydr. (10 LB cellulose)Vdw + Helfr.+ Hydr. (30 LB cellulose)ΠvdW + ΠHelf-700-900-1100

Π(D) = 0 at Deq = 170 Å
Dexp = 200 Å

Π(D) = 0 at Deq = 480 Å
Dexp = 380 Å

Deq agrees well with Dexp, confirming interplays of interfacial forces.
Rossetti, Schneck, Kaufmann, Fragneto, Konovalov, Tanaka, submitted

Modeling Interactions Mediated via “MembraneBound” Sugars (Glycocalyx)
Planar stacks of synthetic/natural glycolipid membranes hydrated in bulk
D2O or vapor at defined ΠD2O and T:
E. Schneck
Multiple Polymer-Supported Membranes
Synthetic Glycolipids

R. Schmidt (Konstanz)
Bacterial Lipopolysaccharides

U. Seydel, K. Brandenburg (Borstel), T. Beverage (Guelph)

Specular/Off-Specular Neutron Scattering

Planar Geometry:

E. Schneck
B. Demé (ILL D16)

Identification of vertical and lateral scattering vector components
2p2pqz =[sin (G - W ) + sin (W )]q|| =[cos(G - W ) - cos(W )]llSafinya, et al. Phys. Rev. Lett. (1987), Salditt, J. Phys. Cond. Matt. (2005)

Specular
Intensity

G = 2W

q|| = 0

Vertical structure, inter-membranepotential

Off-SpecularIntensity

G „ 2Wq|| „ 0Lateral structural ordering, membranemechanics

Simulation of Scattering Signals (1)Basic Framework: Discrete Smectic Hamiltonian
N -12k 2
 B22(u n+1 - u n ) +H = ∫ d r∑ 
xy u nA2

n =1  2 d()

Leibler & Lipowsky, Phys. Rev. B (1987)

Two key parameters: compression modulus B & bending rigidity κScattering from stratified rough interfaces in 1st Born approximation as a function of qz and q||S (q z , q|| )e- q z2s 2q z2¥N
q z2 (s 2 - g k ( r ) 2 ) -iq|| re dr 
 N + 2∑ ( N - k ) cos(kq z d ) ∫ ek =1-¥Sinha, J. Phys. III (1994)

Displacement correlation function gk(r)is determined by two mechanicalparameters: λ & ηlk
B

De Gennes Parameter hg k (r ) =1kB

d2p2

[1 - J (q r )exp(- lkq d )]dq

¥h ∫o2p R

2||||q|| 1 +l2 d 2
4||q||4

Lei, Safinya, Bruinsma, J. Phys. II (1995)

Caillé Parameter

Schneck, Rehfeldt, Oliveira, Gege, Schmidt, Demé, Tanaka, Phys. Rev. E (2008)

Simulation of Scattering Signals (2)2nd Bragg Sheet was taken to ensure 1st Born approx.
ExperimentInt. along ΓModelSheet width along ΩSynthetic glycolipids
in bulk D2O

The strategy is applicable for both synthetic lipids and natural compounds (e.g.
lipopolysaccharides from bacteria mutants) to highlight the influence of molecularchemistry and mutation on structural ordering and mechanics of biomembranes, e.g.
κ(lipid A) = 0.9 MPa but κ(LPSRa) = 1.6 MPa.
Schneck, et al., Phys. Rev. E (2008), Phys. Rev. E (2009), Oliveira, et al., Comptes Rendus Chimie (2009),Schneck et al., Roy. Soc. J. Interf. (2009)

Mechanism of Bacterial Resistance against CAPs
Canadian COE “Advanced Food and Material Network”

In-vivo study (T. Beverage, Univ. Guelph)
Significant (> 5 x) bacterial survival with Ca2+.

Coarse-Grained MC simulation (D. Pink, Canadian COE)
Sugar chains should “collapse” in the presence of Ca2+.
Pink, Truelstrup Hansen, Gill, Quinn, Jericho, Beveradge, Langmuir (2003).

No experimental study has revealed the influence of Ca2+
on the molecular level.

Our Strategy: Design of Simple Models of the Outer
Surface of Bacteria with Well-Defined Building Blocks

Model: Monolayer of Bacterial
LPSs at the air/water interface

Alberts “Molecular Biology of the Cells”

Building Blocks:
Purified LPSs

PAO1 LPS
Beverage Lab / Guelph

LPS ReLPS RaLipid A (core)

Seydel & Brandenburg Lab / Borstel

Vertical Structures: Grazing Incidence X-ray Scattering
Out of Specular Plane (GIXOS) at the Air/Water Interface
ESRF ID10B beam line (O. Konovalov)

λ = 1.547 ǺPenetration depth

αi = 0.119 °a
L = qc 1 -  i   ~ 5nm a c  2-1Detection with a linear PSD at q|| ~ 0.029 Å–1
Oliveira, Schneck, Konovalov, Brandenburg, Seydel, Quinn, Pink, Tanaka
Comptes Rendus,(2009), Phys. Rev. E (2009)

GIXOS: Principles
For conformal layers, I(qz) at q|| ~ 0 is connected to the correspondingreflectivity:

I (q z )T (k out )Vineyard Function

2R(q z )RF (q z )Mora et al., Europhys. Lett. (2004)

To obtain a least square fit to each result, we developed a new fitting
routine based on the Master Formula for specular reflectivity:

1 dr ( z )R(q z ) = R F (q z ) ∫exp(iq z z )dzrdz

2Compared to specular reflectivity (θ-2θ scan), GIXOS can minimize theradiation time (< 100 times) that is advantageous for biological samples.

Influence of Ca2+ on LPSRa
GIXOS at A = 180 Ǻ2

MCElectron density profiles

Collapse of sugar chains in thepresence of Ca2+ could be detected.
Coarse-grained MC simulations
based on linearlized PB theorem
fully supported our experimental
findings.
Oliveira, et al. Comptes Rendus (2009)

Impact of Herring Protamine (Ptm)
Canadian Inst. Fishery

π vs. time at A = 180 Ǻ2
Ca2+-free:∆π ~ 15 mNm–1

Destruction of layered structure
by Ptm intrusion
Ca2+-loaded:
Membrane remains intact!

MC simulation predicts the creation

no Ca2+
with Ca2+of an electrostatic energy barrier (ataround z = 20 Å) repelling positively
charged protamine.
Oliveira, Schneck, Konovalov, Brandenburg, Seydel, Quinn, Pink, Tanaka, Compt. Rendus (2009)

Next Step: Determination of Ion Specific Density Profiles
Grazing Incidence X-ray Fluorescence
Variation of αi (and thus Λ)a
L = qc 1 -  i   a c  2-1

Fluorescence from the target elements (K: 3.31 keV, Ca: 3.69 keV) measured atvarious αi enables us to calculate the depth profiles/amounts of ions instead of thewhole electron density profiles.
Schneck, Schubert, Brandenburg, Konovalov, Quinn, Pink, Tanaka, submitted.

Conclusions• Specular reflectivity at solid/liquid interface of well-definedpolymer-supported membranes enable us to quantify the physicalroles biopolymers in modulating bioloical interfaces.
• The impact of membrane-bound sugars on the cell/cell contacts
can be evaluated with the vertical compressibility and bending
rigidity measured by specular/off-specular scattering of planar
membrane stacks.
• Crucial roles of Ca2+ on the bacterial resistance against
intruders (CAPs) can be revealed by grazing-incidence scattering
at the air/water interface.

Thanks!
Current Members
S. Kaufmann, F. Rossetti , H. Yoshikawa,M. Kazanci, F. Al-Ali, J. Oelke, E.
Schneck, M. Tutus, T. Schubert, P. Seitz,W. Abuillan, T. Kaindl, D. Gassull, H.
Rieger, K. Hock, M. Hermann, J. Oswald,
A. Eichmann, M. Pascuci, L. Wolber, C.
Harasim, …

R. Jordan, M. Stutzmann, M. Eickhoff, G. Abstreiter, M. Tornow, E. Sackmann (München), R. Schmidt(Konstanz), G. Wegner (MPI Mainz), S. Armes (Sheffield), M. Lanzer, T. Holstein, A. Ho (Heidelberg),
A. Gast, S. Boxer (Stanford), U. Seydel (FZ Borstel), S. Funari, (HASYLAB), O. Konovalov (ESRF), B. Demé,G. Fragnetto (ILL), Bayer (Leverkusen), General Mills (Conneticut), Advalytix (München)…

German Science Foundation (DFG), German Excellence
Initiatives, Ministry of Research and Education(BMBF), Helmholtz Society, State Baden-Württenberg,
Heidelberg Academy of Science, Humboldt Foundation,DAAD, EU FP6/7, VDI, Canadian COE etc.