General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.

• Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal

If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.

Downloaded from orbit.dtu.dk on: Jun 14, 2018

Glycoproteins at the rubbing interfaces of biosystems

Lee, Seunghwan

Publication date:2010

Document VersionPublisher's PDF, also known as Version of record

Link back to DTU Orbit

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: seele@mek.dtu.dk

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

Lubricity of mucins/mucus gels

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

Lubrication properties of PGM solution: pH 7

Lubrication properties of PGM solution: pH 2

Lubrication properties of PGM solution: pH 12

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 (Pluronic®)

CH2CH2O CH2CH2OCH2CHO

CH3n nm

PEO‐b‐PPO‐b‐PEO

3

f PEO

% of

M.W. of PPO

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: Adsorption behavior

200

150

g/c

m2)

100

bed

mas

s (n

g

50Adso

rb

2 2 770

1 2

PGM BSM

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)

A proposed model: In bulk solution 

A proposed model: At liquid/solid interface

pH 7pH 7p

tribostress

pH 2pH 2

tribostress

Thank you for your attention!y y