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Glycoproteins at the rubbing interfaces of biosystems
Lee, Seunghwan
Publication date:2010
Document VersionPublisher's PDF, also known as Version of record
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Citation (APA):Lee, S. (2010). Glycoproteins at the rubbing interfaces of biosystems [Sound/Visual production (digital)]. 4thProtein.DTU Workshop, Technical University of Denmark, 01/01/2010
Glycoproteins at the Rubbing Interfaces of BiosystemsInterfaces of Biosystems
4th Workshop in Proteins.DTUNovember 12, 2010, DTU
Seunghwan LeeDepartment of Mechanical Engineering, DTU
Contact: [email protected]
Water as a lubricant
Oil
Nature’s primary choice of lubricant
Men’s primary choice of lubricant
Water
lubricantlubricant
Challenges in oil‐based lubrication:f h til l 0 001!
g
limited resources
environmental issue (especially additives)
µ for human cartilage: as low as 0.001!
Water as a lubricant in engineering point of view
‐ non‐toxic
‐ environmentally‐friendly
readily available and cost effective
‐ poor pressure response
low pressure‐coefficient of viscosity‐ readily available and cost effective
‐ non‐flammable
‐ high thermal capacity
p ywater: α = 0.36 GPa‐1
oil: α = 10‐20 GPa‐1
limited application temperature‐ biocompatible
‐ limited application temperature
‐ corrosion for ferrous materials
water oil
Nature’s approach to use water as lubricant
brush‐like, sugar‐basedmacromolecules
Mucins
Mucus (gel)Mucin (polymer)
P t l t
PGM (STM, 360 nm × 360 nm)
Roberts, CJ et al Proteins and Proteoglycan aggregate
• plays a key structural role in cartilage
Peptide Letters 1995 2, 409
Lubricin
• mucinous glycoprotein of the synovialsugar chains
link protein
hyaluronan
core protein
g y p yfluid (250 µg/ml, MW = 2.3 × 105 g/mol)
S. Lee et al., SCIENCE 2008
y
Mucus, Mucin, and Mucin Domains
stiff , charged, hydrophilic, ca. 70% of mass
Schematic representation of the mucin
mucus (gel)2HN COOH
maybe flexible, charged, hydrophobic/‐philic
mucus (gel)
Water
Salts
IgG
polypeptide
Proteins
mucins
mucin (polymer)
Schematic representation of the Lubricin (PRG 4)
mucin (polymer)
Zappone B et al, Langmuir 2008, 24, 1495.
Roberts, CJ et al, Proteins and Peptide Letters 1995, 2, 409
PGM, STM (360 nm � 360 nm)
Mucus gels
Hattrup CL and Gendler SJ, Ann. Rev. Physol. 2008, 70, 431
S‐S S‐S
Gel‐formation (in vivo)disulfide bonding
hydrophobic interaction sugar‐sugar interaction
Monolayer of mucins at water/solid interface
(sub)monolayer surface‐coating(sub)monolayer surface‐coatingwater
solid
Hydrophilicity
L. Shi and K.D. Caldwell, J. Colloid & Interf. Sci. (2000) 224, 372‐381
Suppression of proteins and bacteria adsorptionSuppression of proteins and bacteria adsorption L. Shi, R. Ardehali, P. Valint, & K.D. Caldwell, Biotech. Letters (2001) 23, 437‐441
LubricationI.C.H. Berg, L. Lindh & T. Arnebrant, Biofouling (2004) 20, 65‐70
S. Lee, M. Müller, K. Rezwan, N.D. Spencer, Langmuir (2005) 21, 8344‐8353
h h l l
2HN COOH
Mucins as a amphiphilic copolymer
‐CH2CH2O‐ ‐CH2CH2O‐‐CH2CHO‐
m mnCH3
m mn
Model surface and pin‐on‐disk tribometry
pin‐on‐disk tribometerElastomer as model surface of biological i h l
loaddead
tissues: mimic mechanical properties
lever
Pin (PDMS)
deadweight
PGM-containingYoung´s modulus
Poly(dimethylsiloxane) (PDMS)
aqueous solutionYoung s modulus
ca. 2 MPa
Poission ratio
rotationalmotion
disk (PDMS)
L d 1 N
Poission ratio
0.5
side view
Load = 1 N
P ~ 0.5 MPa
Optical Waveguide Lightmode Spectroscopy (OWLS)
Surface adsorption properties: OWLS
Optical Waveguide Lightmode Spectroscopy (OWLS)
Adsorption of mucins onto PDMS surface dso pt o o uc s o to S su ace
*PDMS (~30nm)
Waveguide(SiOx0.75TiOx0.25)
TM TE
incidence angle
Surface adsorption properties: OWLS
300
250
PGM ( H 2)m2 )
150
200 PGM (pH 2)PGM (pH 7)PGM (pH 12)
mas
s (n
g/cm
100
Adso
rbed
m
0
50
0 5 10 15 20 25 30 35 40
buffer rinsingA
0 5 10 15 20 25 30 35 40time (minutes)
PGM, 1mg/ml buffer: 1mM KH2PO4, KCl 0.1M
Surface adsorption properties: OWLS
pH and ionic strength dependence
300pH 2pH 7
H 12ng/c
m2 )
200
pH 12
d m
ass
(n
100
Ads
orbe
d
00 0001 0 001 0 01 0 1 1 10
A
0.0001 0.001 0.01 0.1 1 10
total ionic strength (M)
Tertiary structure: Near‐UV CD spectroscopy
pH dependence
20
30pH2pH4pH7
pH 7
0
10pH10pH12
-20
-10pH 2
-30250 270 290 310 330 350
wavelength (nm)
disruption of tertiary structure of “naked” polypeptide region
a e e g ( )
PGM, 1mg/ml buffer: 1mM KH2PO4, KCl 0.1M
Tertiary structure: Near‐UV CD spectroscopy
Ionic strength dependence
20
30pH2 (buffer only)pH2 (KCl, 0.01M)pH2 (KCl, 0.1M)pH2 (KCl, 1.0M)
0
10
p ( )pH7 (buffer only)pH7 (KCl, 0.01M)pH7 (KCl, 0.1M)pH7 (KCl, 1.0M)pH12 (buffer only)pH12 (KCl, 0.01M)
-20
-10pH12 (KCl, 0.1M)pH12 (KCl, 1.0M)
-30250 270 290 310 330 350
wavelength (nm)a e e g ( )
PGM, 1mg/ml buffer: 1mM KH2PO4, KCl 0.1M
A schematic model at the sliding interface
Before sliding After sliding
1 turnpH 71
n (N
)pH 7
0,5frict
ion
pH 12
00 1250
rotation (or accumulated scan length) pH 2
pH 2
Model polysaccharides : Dextran and Hyaluronic acid
1
10 Lubrication
0 1
1
μbuffer (pH7)buffer (pH2)
Dextran
Hyaluronic acid 0.01
0.1 buffer (pH2)dextran (pH7)dextran (pH2)HA (pH7)HA (pH2)
0.011 10 100
speed (mm/sec)
Adsorption
200
250
300
Hyaluronic AcidDextran
Adsorption
0
100
150
0
50
0 5 10 15 20 25 30time (min)
Model proteins : albumin
10 pH 7 (buffer)10n
pH 7pH 2 (buffer)pH 2
1
t of f
rictio
n
0.1
Coe
ffici
en
0 010.011 10 100
Velocty (mm/sec)
buffer = KH2PO4 10 mMLoad : 1 N
H k D E f h i
Soft Elastohydrodynamic Lubrication (soft EHL)
Hamrock, Dowson, EsfahanianHamrock, B.J. and Dowson, D., Proc. 5th Leeds‐Lyon symp. on Trib. 22‐27 (1979)
Esfahanian, M. and Hamrock, B.J., Tribol. Trans. 34, 628‐632 (1991)
H d EHL h 0 47 0 49 0 68 0 68 0 12 0 07Hard EHL
Soft EHL
hmin = 1.79 R0.47α 0.49 η0 0.68 U0.68 E‐0.12 W‐0.07
hmin = 2.8 R0. η0 0.65 U0.65 E‐0..44 W‐0.21
α : pressure coefficient of viscosity
rigidrigid elastic
300
R = 3 mm
1 N Soft contact: PDMS vs. PDMS
Rigid contact: steel vs. steel 200
250"soft" contacts
"rigid" contacts
ess (
nm)
E (PDMS) = 2 MPa
ν (PDMS) = 0.5
50
100
150fil
m th
ickn
e
E (steel) = 200 GPa
ν (steel) = 0.3 0
50
0.0001 0.001 0.01 0.1 1
f
speed (m/s)
1010
Effect of surface hydrophilicity
PDMS
PDMS1
10
1
10
0.1μ O2 plasma
ox‐PDMS
0.1μ
0.010.1 1 10 100
speed (mm/sec)
ox‐PDMS0.01
0.1 1 10 100speed (mm/sec)
No significant change in bulkNo significant change in bulk mechanical properties
Hydrophilization of surface ( OH and/or COOH groups)(‐OH and/or ‐COOH groups)
PEO‐b‐PPO‐b‐PEO
Adsorption of albumin
1
10
0.1
1μ
bufferF68P105
0.010.001 0.01 0.1
speed (m/sec)J. Biomed. Mat. Res., 1998, R.J. Green et al
speed (m/sec)
F68 EO76.4 PO29 EO76.4
tribostress
P105 EO PO EO
tribostress
P105 EO36.9 PO56 EO36.9
Mucins from different organs: similarity and difference
pH 2pH 7
PDMS
PDMS
PDMS
PDMS
Bansil et al, Annu. Rev. Physiol. 1995, 57, 635.
10PGM
(Porcine Gastric Mucin)
1010
111pH 7 pH 2
0.1
μ BSM(Bovine Submaxillary Mucin)
0.1
μ0.1
μ
0 010 010 01
pH 7 pH 2
0.010.1 1 10 100
sliding speed (mm/s) S. Lee et al., unpublished
0.010.1 1 10 100
sliding speed (mm/s)
0.010.1 1 10 100
sliding speed (mm/s)
BSM vs. PGM: Size (Dynamic Light Scattering)
30pH 7
pH 2
pH 7
10
20 pH 2
01 10 100 1000 10000
diameter (nm)diameter (nm)
1
2
Increase in size (aggregation) at pH 2 is general for both mucins,
0 50 100 150 200Z-average (nm)
pH 2 is general for both mucins, but is more pronounced for PGMthan BSM
PGM (pH 2) PGM (pH 7)UV/Vis spectroscopy
BSM vs. PGM: Protein composition
4
52.5
PGMε
BSM (pH 2) BSM (pH 7) UV/Vis spectroscopy
3
41
1.5
2
Inte
nsity
(a.u
.)
BSMε280
2
30
0.5
1 2
pH 7 pH 2
1
2
0200 250 300 350 400 450 500
wavelength (nm)
Tertiary structure: Near UV CDTertiary structure: Near UV CDSecondary structure: Far UV CDSecondary structure: Far UV CD
BSM vs. PGM: Protein conformation
Tertiary structure: Near‐UV CD Tertiary structure: Near‐UV CD
20
30Secondary structure: Far‐UV CD Secondary structure: Far‐UV CD
40
60
-10
0
10
-20
0
20
-30
-20
250 300 350-60
-40
200 210 220 230 240 250 250 300 350wavelength (nm)
200 210 220 230 240 250
wavelength (nm)
300Protein unfolding: Fluorescence Spec.Protein unfolding: Fluorescence Spec.
150
200
250
50
100
150
0300 320 340 360 380 400 420 440
wavelength (nm)