Downloaded from orbit.dtu.dk on: Dec 19, 2018 Glycoproteins at the rubbing interfaces of biosystems Lee, Seunghwan Publication date: 2010 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Lee, S. (Invited author). (2010). Glycoproteins at the rubbing interfaces of biosystems. Sound/Visual production (digital) 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.
Glycoproteins at the Rubbing Interfaces of Biosystems 4th Workshop in Proteins.DTU November 12, 2010, DTU Seunghwan Lee Department of Mechanical Engineering, DTU Contact: seele@mek.dtu.dk
Water as a lubricant Oil Water Men s primary choice of lubricant Nature s primary choice of lubricant Challenges in oil based lubrication: limited resources environmental issue (especially additives) µ for human cartilage: as low as 0.001! 001!
Water as a lubricant in engineering point of view non toxic environmentally friendly readilyavailableand available and cost effective non flammable high thermal capacity biocompatible poor pressure response low pressure coefficient of viscosity water: α = 0.36 GPa 1 oil: α = 10 20 GPa 1 limited application temperature corrosion for ferrous materials water oil
Nature s approach to use water as lubricant brush like, sugar based macromolecules Mucins Mucus (gel) Mucin (polymer) Proteoglycan aggregate plays a key structural role in cartilage Lubricin PGM (STM, 360 nm 360 nm) Roberts, CJ et al Proteins and Peptide Letters 1995 2, 409 sugar chains link protein core protein hyaluronan mucinous glycoprotein gy of the synovial fluid (250 µg/ml, MW = 2.3 10 5 g/mol) S. Lee et al., SCIENCE 2008
Lubricity of mucins/mucus gels
Mucus, Mucin, and Mucin Domains Schematic representation of the mucin stiff, charged, hydrophilic, ca. 70% of mass mucus (gel) mucin (polymer) Water Salts IgG Proteins mucins 2HN Schematic representation of the Lubricin (PRG 4) maybe flexible, charged, hydrophobic/ philic polypeptide COOH Zappone B et al, Langmuir 2008, 24, 1495. PGM, STM (360 nm 360 nm) Roberts, CJ et al, Proteins and Peptide Letters 1995, 2, 409
Mucus gels Hattrup CL and Gendler SJ, Ann. Rev. Physol. 2008, 70, 431 Gel formation (in vivo) S S disulfide bonding S S hydrophobic interaction sugar sugar interaction
Monolayer of mucins at water/solid interface water solid (sub)monolayer surface coating Hydrophilicity L. Shi and K.D. Caldwell, J. Colloid & Interf. Sci. (2000) 224, 372 381 Suppression of proteins and bacteria adsorption Lubrication L. Shi, R. Ardehali, P. Valint, & K.D. Caldwell, Biotech. Letters (2001) 23, 437 441 I.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 Mucins as a amphiphilic hl copolymer 2HN COOH CH 2 CH 2 O CH 2 CHO CH 2 CH 2 O m CH 3 n m
Model surface and pin on disk tribometry Elastomer as model surface of biological tissues: mimic mechanical properties pin on disk tribometer Poly(dimethylsiloxane) (PDMS) dead weight load lever Pin (PDMS) Young s modulus PGM-containing aqueous solution ca. 2 MPa Load = 1 N Poission ratio 0.5 rotational motion disk (PDMS) P ~ 0.5 MPa side view
Lubrication properties of PGM solution: ph 7
Lubrication properties of PGM solution: ph 2
Lubrication properties of PGM solution: ph 12
Surface adsorption properties: OWLS Optical Waveguide Lightmode Spectroscopy (OWLS) Adsorption o of mucins onto PDMS surface *PDMS (~30nm) Waveguide (SiOx 0.75 TiOx 0.25 ) TM TE incidence angle
Surface adsorption properties: OWLS 300 Adsorbed mass (ng/cm 2 ) 250 200 150 100 50 PGM (ph 2) PGM (ph 7) PGM (ph 12) buffer rinsing 0 0 5 10 15 20 25 30 35 40 time (minutes) PGM, 1mg/ml buffer: 1mM KH 2 PO 4, KCl 0.1M
Surface adsorption properties: OWLS ph and ionic strength dependence Adsorbed d mass (n ng/cm 2 ) 300 200 100 ph 2 ph 7 ph H12 0 0.00010001 0.001001 001 0.01 01 0.1 1 10 total ionic strength (M)
Tertiary structure: Near UV CD spectroscopy ph dependence 30 20 10 ph2 ph4 ph7 ph10 ph12 ph 7 0-10 ph 2-20 -30 250 270 290 310 330 350 wavelength aeeg (nm) disruption of tertiary structure of naked polypeptide region PGM, 1mg/ml buffer: 1mM KH 2 PO 4, KCl 0.1M
Tertiary structure: Near UV CD spectroscopy Ionic strength dependence 30 20 10 0-10 ph2 (buffer only) ph2 (KCl, 0.01M) ph2 (KCl, 0.1M) ph2 (KCl, 1.0M) ph7 (buffer only) ph7 (KCl, 0.01M) ph7 (KCl, 0.1M) ph7 (KCl, 1.0M) ph12 (buffer only) ph12 (KCl, 0.01M) ph12 (KCl, 0.1M) ph12 (KCl, 1.0M) -20-30 250 270 290 310 330 350 wavelength aeeg (nm) PGM, 1mg/ml buffer: 1mM KH 2 PO 4, KCl 0.1M
A schematic model at the sliding interface Before sliding After sliding ph 7 1 1 turn ph 7 friction (N) 0,5 ph 2 ph 12 0 0 1250 rotation (or accumulated scan length) ph 2
Model polysaccharides : Dextran and Hyaluronic acid 10 Lubrication Dextran Hyaluronic acid μ 1 buffer (ph7) 01 0.1 buffer (ph2) dextran (ph7) dextran (ph2) HA (ph7) HA (ph2) 0.01 1 10 100 speed (mm/sec) 300 Adsorption 250 Hyaluronic Acid Dextran 200 150 100 50 0 0 5 10 15 20 25 30 time (min)
Model proteins : albumin 10 ph 7 (buffer) ph 7 ph 2 (buffer) ph 2 t of friction 1 Coefficien 0.1 001 0.01 1 10 100 Velocty (mm/sec) Load : 1 N buffer = KH 2 PO 4 10 mm
Soft Elastohydrodynamic Lubrication (soft EHL) Hamrock, Dowson, Efh Esfahanian Hamrock, 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) Hard dehl h 047 049 068 068 012 007 min = 1.79 R 0.47 α 0.49 η 0.68 0 U 0.68 E 0.12 W 0.07 Soft EHL h min = 2.8 R 0. η 0 0.65 U 0.65 E 0..44 W 0.21 α : pressure coefficient of viscosity rigid elastic 1 N Soft contact: PDMS vs. PDMS R = 3 mm film thickne ess (nm) 300 250 Rigid contact: steel vs. steel 200 150 E(PDMS)= 2 MPa ν (PDMS)= 0.5 100 50 E (steel) = 200 GPa ν (steel) = 0.3 0 "soft" contacts "rigid" contacts 0.0001 0.001 0.01 0.1 1 speed (m/s)
Effect of surface hydrophilicity μ 10 1 0.1 PDMS PDMS O 2 plasma 0.01 0.1 1 10 100 speed (mm/sec) ox PDMS ox PDMS No significant change in bulk mechanical properties Hydrophilization of surface ( OH and/or COOH groups)
PEO b PPO b PEO (Pluronic ) CH 2 CH 2 O CH 2 CHO CH 2 CH 2 O n CH m 3 PEO b PPO b PEO n f PEO % of M.W. of PPO
PEO b PPO b PEO Adsorption of albumin 10 μ 1 0.1 buffer F68 P105 J. Biomed. Mat. Res., 1998, R.J. Green et al 0.01 0.001 0.01 0.1 speed (m/sec) tribostress F68 EO 76.4 PO 29 EO 76.4 P105 EO 36.9 PO 56 EO 36.9 tribostress
Mucins from different organs: similarity and difference ph 7 ph 2 PDMS PDMS Bansil et al, Annu. Rev. Physiol. 1995, 57, 635. PDMS PDMS 10 PGM (Porcine Gastric Mucin) μ 1 0.1 ph 7 ph 2 BSM (Bovine Submaxillary Mucin) 001 0.01 0.1 1 10 100 sliding speed (mm/s) ph 7 ph 2 S. Lee et al., unpublished
BSM vs. PGM: Adsorption behavior 200 Adsorb bed mass (ng g/cm 2 ) 150 100 50 0 2 7 2 7 1 2 PGM BSM
BSM vs. PGM: Size (Dynamic Light Scattering) 30 ph 7 ph 2 20 ph 7 ph 2 10 0 1 10 100 1000 10000 diameter (nm) 2 1 0 50 100 150 200 Z-average (nm) Increase in size (aggregation) at ph 2 is general for both mucins, but is more pronounced for PGM than BSM
BSM vs. PGM: Protein composition 5 PGM (ph 2) PGM (ph 7) BSM (ph 2) BSM (ph 7) UV/Vis spectroscopy 4 3 Intensity (a.u.) 2.5 2 1.5 1 0.5 ε 280 PGM BSM 2 0 1 2 ph 7 ph 2 1 0 200 250 300 350 400 450 500 wavelength (nm)
BSM vs. PGM: Protein conformation 60 Secondarystructure: Far UV CD 30 Tertiary structure: Near UV CD 40 20 20 10 0 0-20 -10-40 -20-60 -30 200 210 220 230 240 250 250 300 350 wavelength (nm) wavelength (nm) 300 250 200 150 100 50 Protein unfolding: Fluorescence Spec. 0 300 320 340 360 380 400 420 440 wavelength (nm)
A proposed model: In bulk solution
A proposed model: At liquid/solid interface ph 7 tribostress ph 7 ph 2 tribostress ph 2
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