Glycoproteins at the rubbing interfaces of biosystems

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1 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.

2 Glycoproteins at the Rubbing Interfaces of Biosystems 4th Workshop in Proteins.DTU November 12, 2010, DTU Seunghwan Lee Department of Mechanical Engineering, DTU Contact:

3 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!

4 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 = GPa 1 limited application temperature corrosion for ferrous materials water oil

5 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 , 409 sugar chains link protein core protein hyaluronan mucinous glycoprotein gy of the synovial fluid (250 µg/ml, MW = g/mol) S. Lee et al., SCIENCE 2008

6 Lubricity of mucins/mucus gels

7 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, PGM, STM (360 nm 360 nm) Roberts, CJ et al, Proteins and Peptide Letters 1995, 2, 409

8 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

9 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, Suppression of proteins and bacteria adsorption Lubrication L. Shi, R. Ardehali, P. Valint, & K.D. Caldwell, Biotech. Letters (2001) 23, I.C.H. Berg, L. Lindh & T. Arnebrant, Biofouling (2004) 20, S. Lee, M. Müller, K. Rezwan, N.D. Spencer, Langmuir (2005) 21, 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

10 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

11 Lubrication properties of PGM solution: ph 7

12 Lubrication properties of PGM solution: ph 2

13 Lubrication properties of PGM solution: ph 12

14 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

15 Surface adsorption properties: OWLS 300 Adsorbed mass (ng/cm 2 ) PGM (ph 2) PGM (ph 7) PGM (ph 12) buffer rinsing time (minutes) PGM, 1mg/ml buffer: 1mM KH 2 PO 4, KCl 0.1M

16 Surface adsorption properties: OWLS ph and ionic strength dependence Adsorbed d mass (n ng/cm 2 ) ph 2 ph 7 ph H total ionic strength (M)

17 Tertiary structure: Near UV CD spectroscopy ph dependence ph2 ph4 ph7 ph10 ph12 ph ph wavelength aeeg (nm) disruption of tertiary structure of naked polypeptide region PGM, 1mg/ml buffer: 1mM KH 2 PO 4, KCl 0.1M

18 Tertiary structure: Near UV CD spectroscopy Ionic strength dependence 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) wavelength aeeg (nm) PGM, 1mg/ml buffer: 1mM KH 2 PO 4, KCl 0.1M

19 A schematic model at the sliding interface Before sliding After sliding ph turn ph 7 friction (N) 0,5 ph 2 ph rotation (or accumulated scan length) ph 2

20 Model polysaccharides : Dextran and Hyaluronic acid 10 Lubrication Dextran Hyaluronic acid μ 1 buffer (ph7) buffer (ph2) dextran (ph7) dextran (ph2) HA (ph7) HA (ph2) speed (mm/sec) 300 Adsorption 250 Hyaluronic Acid Dextran time (min)

21 Model proteins : albumin 10 ph 7 (buffer) ph 7 ph 2 (buffer) ph 2 t of friction 1 Coefficien Velocty (mm/sec) Load : 1 N buffer = KH 2 PO 4 10 mm

22 Soft Elastohydrodynamic Lubrication (soft EHL) Hamrock, Dowson, Efh Esfahanian Hamrock, B.J. and Dowson, D., Proc. 5th Leeds Lyon symp. on Trib (1979) Esfahanian, M. and Hamrock, B.J., Tribol. Trans. 34, (1991) Hard dehl h min = 1.79 R 0.47 α 0.49 η U 0.68 E 0.12 W 0.07 Soft EHL h min = 2.8 R 0. η U 0.65 E W 0.21 α : pressure coefficient of viscosity rigid elastic 1 N Soft contact: PDMS vs. PDMS R = 3 mm film thickne ess (nm) Rigid contact: steel vs. steel E(PDMS)= 2 MPa ν (PDMS)= E (steel) = 200 GPa ν (steel) = "soft" contacts "rigid" contacts speed (m/s)

23 Effect of surface hydrophilicity μ PDMS PDMS O 2 plasma speed (mm/sec) ox PDMS ox PDMS No significant change in bulk mechanical properties Hydrophilization of surface ( OH and/or COOH groups)

24 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

25 PEO b PPO b PEO Adsorption of albumin 10 μ buffer F68 P105 J. Biomed. Mat. Res., 1998, R.J. Green et al speed (m/sec) tribostress F68 EO 76.4 PO 29 EO 76.4 P105 EO 36.9 PO 56 EO 36.9 tribostress

26 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 ph 7 ph 2 BSM (Bovine Submaxillary Mucin) sliding speed (mm/s) ph 7 ph 2 S. Lee et al., unpublished

27 BSM vs. PGM: Adsorption behavior 200 Adsorb bed mass (ng g/cm 2 ) PGM BSM

28 BSM vs. PGM: Size (Dynamic Light Scattering) 30 ph 7 ph 2 20 ph 7 ph diameter (nm) Z-average (nm) Increase in size (aggregation) at ph 2 is general for both mucins, but is more pronounced for PGM than BSM

29 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.) ε 280 PGM BSM ph 7 ph wavelength (nm)

30 BSM vs. PGM: Protein conformation 60 Secondarystructure: Far UV CD 30 Tertiary structure: Near UV CD wavelength (nm) wavelength (nm) Protein unfolding: Fluorescence Spec wavelength (nm)

31 A proposed model: In bulk solution

32 A proposed model: At liquid/solid interface ph 7 tribostress ph 7 ph 2 tribostress ph 2

33 Thank you for your attention!